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Journal articles on the topic 'Substrate specificity'

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Author: Grafiati

Published: 4 June 2021

Last updated: 11 March 2023

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1

Koudelakova, Tana, Eva Chovancova, Jan Brezovsky, Marta Monincova, Andrea Fortova, Jiri Jarkovsky, and Jiri Damborsky. "Substrate specificity of haloalkane dehalogenases." Biochemical Journal 435, no. 2 (March 29, 2011): 345–54. http://dx.doi.org/10.1042/bj20101405.

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An enzyme's substrate specificity is one of its most important characteristics. The quantitative comparison of broad-specificity enzymes requires the selection of a homogenous set of substrates for experimental testing, determination of substrate-specificity data and analysis using multivariate statistics. We describe a systematic analysis of the substrate specificities of nine wild-type and four engineered haloalkane dehalogenases. The enzymes were characterized experimentally using a set of 30 substrates selected using statistical experimental design from a set of nearly 200 halogenated compounds. Analysis of the activity data showed that the most universally useful substrates in the assessment of haloalkane dehalogenase activity are 1-bromobutane, 1-iodopropane, 1-iodobutane, 1,2-dibromoethane and 4-bromobutanenitrile. Functional relationships among the enzymes were explored using principal component analysis. Analysis of the untransformed specific activity data revealed that the overall activity of wild-type haloalkane dehalogenases decreases in the following order: LinB~DbjA>DhlA~DhaA~DbeA~DmbA>DatA~DmbC~DrbA. After transforming the data, we were able to classify haloalkane dehalogenases into four SSGs (substrate-specificity groups). These functional groups are clearly distinct from the evolutionary subfamilies, suggesting that phylogenetic analysis cannot be used to predict the substrate specificity of individual haloalkane dehalogenases. Structural and functional comparisons of wild-type and mutant enzymes revealed that the architecture of the active site and the main access tunnel significantly influences the substrate specificity of these enzymes, but is not its only determinant. The identification of other structural determinants of the substrate specificity remains a challenge for further research on haloalkane dehalogenases.

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2

Wilson, Charles, and David A. Agard. "Engineering substrate specificity." Current Opinion in Structural Biology 1, no. 4 (August 1991): 617–23. http://dx.doi.org/10.1016/s0959-440x(05)80086-7.

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3

Wong, W. "Specifying Substrate Specificity." Science Signaling 6, no. 280 (June 18, 2013): ec140-ec140. http://dx.doi.org/10.1126/scisignal.2004423.

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4

Houlston, C. E., M. Cummings, H. Lindsay, S. Pradhan, and R. L. P. Adams. "DNA substrate specificity of pea DNA methylase." Biochemical Journal 293, no. 3 (August 1, 1993): 617–24. http://dx.doi.org/10.1042/bj2930617.

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DNA methylase, present in low-salt extracts of nuclei prepared from Pisum sativum shoot tips, methylates model DNA substrates containing CNG trinucleotides or CI dinucleotides only. The binding to the hemimethylated trinucleotide substrates is very much stronger and more persistent than the binding to the unmethylated substrates or to the hemimethylated dinucleotide substrate. When the DNA concentration is limiting, the rate of methyl-group transfer with the hemimethylated CNG substrate is much greater than that with the unmethylated CNG. However, the Vmax. is similar for the two CNG substrates. On fractionation using Q-Sepharose, two peaks of activity are seen with different relative activities using the di- and trinucleotide substrates. The relative activity with these substrates changes during purification, during plant growth and on heating at 35 degrees C as well, indicating that more than one enzyme or more than one form of the enzyme may be present.

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5

LINDSTAD, I. Rune, Peter KÖLL, and John S. McKINLEY-McKEE. "Substrate specificity of sheep liver sorbitol dehydrogenase." Biochemical Journal 330, no. 1 (February 15, 1998): 479–87. http://dx.doi.org/10.1042/bj3300479.

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The substrate specificity of sheep liver sorbitol dehydrogenase has been studied by steady-state kinetics over the range pH 7-10. Sorbitol dehydrogenase stereo-selectively catalyses the reversible NAD-linked oxidation of various polyols and other secondary alcohols into their corresponding ketones. The kinetic constants are given for various novel polyol substrates, including L-glucitol, L-mannitol, L-altritol, D-altritol, D-iditol and eight heptitols, as well as for many aliphatic and aromatic alcohols. The maximum velocities (kcat) and the substrate specificity-constants (kcat/Km) are positively correlated with increasing pH. The enzyme-catalysed reactions occur by a compulsory ordered kinetic mechanism with the coenzyme as the first, or leading, substrate. With many substrates, the rate-limiting step for the overall reaction is the enzyme-NADH product dissociation. However, with several substrates there is a transition to a mechanism with partial rate-limitation at the ternary complex level, especially at low pH. The kinetic data enable the elucidation of new empirical rules for the substrate specificity of sorbitol dehydrogenase. The specificity-constants for polyol oxidation vary as a function of substrate configuration with D-xylo > d-ribo > L-xylo > d-lyxo ≈ l-arabino > D-arabino > l-lyxo. Catalytic activity with a polyol or an aromatic substrate and various 1-deoxy derivatives thereof varies with -CH2OH >-CH2NH2 >-CH2OCH3 ≈-CH3. The presence of a hydroxyl group at each of the remaining chiral centres of a polyol, apart from the reactive C2, is also nonessential for productive ternary complex formation and catalysis. A predominantly nonpolar enzymic epitope appears to constitute an important structural determinant for the substrate specificity of sorbitol dehydrogenase. The existence of two distinct substrate binding regions in the enzyme active site, along with that of the catalytic zinc, is suggested to account for the lack of stereospecificity at C2 in some polyols.

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6

Ananvoranich, Sirinart, and Jean-Pierre Perreault. "Substrate Specificity ofδRibozyme Cleavage." Journal of Biological Chemistry 273, no. 21 (May 22, 1998): 13182–88. http://dx.doi.org/10.1074/jbc.273.21.13182.

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7

Crans, Debbie C., and George M. Whitesides. "Glycerol kinase: substrate specificity." Journal of the American Chemical Society 107, no. 24 (November 1985): 7008–18. http://dx.doi.org/10.1021/ja00310a044.

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8

Capecchi, John T., and Gordon Marc Loudon. "Substrate specificity of pyroglutamylaminopeptidase." Journal of Medicinal Chemistry 28, no. 1 (January 1985): 140–43. http://dx.doi.org/10.1021/jm00379a024.

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9

Ratnikov, Boris I., Piotr Cieplak, Albert G. Remacle, Elise Nguyen, and Jeffrey W. Smith. "Quantitative profiling of protease specificity." PLOS Computational Biology 17, no. 2 (February 22, 2021): e1008101. http://dx.doi.org/10.1371/journal.pcbi.1008101.

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Proteases are an important class of enzymes, whose activity is central to many physiologic and pathologic processes. Detailed knowledge of protease specificity is key to understanding their function. Although many methods have been developed to profile specificities of proteases, few have the diversity and quantitative grasp necessary to fully define specificity of a protease, both in terms of substrate numbers and their catalytic efficiencies. We have developed a concept of “selectome”; the set of substrate amino acid sequences that uniquely represent the specificity of a protease. We applied it to two closely related members of the Matrixin family–MMP-2 and MMP-9 by using substrate phage display coupled with Next Generation Sequencing and information theory-based data analysis. We have also derived a quantitative measure of substrate specificity, which accounts for both the number of substrates and their relative catalytic efficiencies. Using these advances greatly facilitates elucidation of substrate selectivity between closely related members of a protease family. The study also provides insight into the degree to which the catalytic cleft defines substrate recognition, thus providing basis for overcoming two of the major challenges in the field of proteolysis: 1) development of highly selective activity probes for studying proteases with overlapping specificities, and 2) distinguishing targeted proteolysis from bystander proteolytic events.

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10

Hatanaka, Akikazu, Tadahiko Kajiwara, Kenji Matsui, and Hiromitsu Toyota. "Substrate Specificity of Tea Leaf Hydroperoxide Lyase." Zeitschrift für Naturforschung C 47, no. 9-10 (October 1, 1992): 677–79. http://dx.doi.org/10.1515/znc-1992-9-1006.

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Substrate specificity of tea leaf fatty acid hydroperoxide lyase was systematically investigated using an entire series of ω 6-(5)-hydroperoxy-C14-C24 dienoic and trienoic acids as substrates. Unexpectedly, the hydroperoxides of C22 but not natural substrates, i.e., those of C18, showed the highest reactivities for the lyase. The reactivities of the hydroperoxides of trienoic acids were always four to ten times higher than those of the dienoic acids.

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11

DEMENDI, Melinda, Noboru ISHIYAMA, Joseph S. LAM, Albert M. BERGHUIS, and Carole CREUZENET. "Towards a better understanding of the substrate specificity of the UDP-N-acetylglucosamine C4 epimerase WbpP." Biochemical Journal 389, no. 1 (June 21, 2005): 173–80. http://dx.doi.org/10.1042/bj20050263.

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WbpP is the only genuine UDP-GlcNAc (UDP-N-acetylglucosamine) C4 epimerase for which both biochemical and structural data are available. This represents a golden opportunity to elucidate the molecular basis for its specificity for N-acetylated substrates. Based on the comparison of the substrate binding site of WbpP with that of other C4 epimerases that convert preferentially non-acetylated substrates, or that are able to convert both acetylated and non-acetylated substrates equally well, specific residues of WbpP were mutated, and the substrate specificity of the mutants was determined by direct biochemical assays and kinetic analyses. Most of the mutations tested were anticipated to trigger a significant switch in substrate specificity, mostly towards a preference for non-acetylated substrates. However, only one of the mutations (A209H) had the expected effect, and most others resulted in enhanced specificity of WbpP for N-acetylated substrates (Q201E, G102K, Q201E/G102K, A209N and S143A). One mutation (S144K) totally abolished enzyme activity. These data indicate that, although all residues targeted in the present study turned out to be important for catalysis, determinants of substrate specificity are not confined to the substrate-binding pocket and that longer range interactions are essential in allowing proper positioning of various ligands in the binding pocket. Hence prediction or engineering of substrate specificity solely based on sequence analysis, or even on modelling of the binding pocket, might lead to incorrect functional assignments.

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12

Sorimachi, Hiroyuki, Hiroshi Mamitsuka, and Yasuko Ono. "Understanding the substrate specificity of conventional calpains." Biological Chemistry 393, no. 9 (September 1, 2012): 853–71. http://dx.doi.org/10.1515/hsz-2012-0143.

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Abstract: Calpains are intracellular Ca2+-dependent Cys proteases that play important roles in a wide range of biological phenomena via the limited proteolysis of their substrates. Genetic defects in calpain genes cause lethality and/or functional deficits in many organisms, including humans. Despite their biological importance, the mechanisms underlying the action of calpains, particularly of their substrate specificities, remain largely unknown. Studies show that certain sequence preferences influence calpain substrate recognition, and some properties of amino acids have been related successfully to substrate specificity and to the calpains’ 3D structure. The full spectrum of this substrate specificity, however, has not been clarified using standard sequence analysis algorithms, e.g., the position-specific scoring-matrix method. More advanced bioinformatics techniques were used recently to identify the substrate specificities of calpains and to develop a predictor for calpain cleavage sites, demonstrating the potential of combining empirical data acquisition and machine learning. This review discusses the calpains’ substrate specificities, introducing the benefits of bioinformatics applications. In conclusion, machine learning has led to the development of useful predictors for calpain cleavage sites, although the accuracy of the predictions still needs improvement. Machine learning has also elucidated information about the properties of calpains’ substrate specificities, including a preference for sequences over secondary structures and the existence of a substrate specificity difference between two similar conventional calpains, which has never been indicated biochemically.

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13

Lamort, Anne-Sophie, Rodolphe Gravier, Anni Laffitte, Luiz Juliano, Marie-Louise Zani, and Thierry Moreau. "New insights into the substrate specificity of macrophage elastase MMP-12." Biological Chemistry 397, no. 5 (May 1, 2016): 469–84. http://dx.doi.org/10.1515/hsz-2015-0254.

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Abstract Macrophage elastase, or MMP-12, is mainly produced by alveolar macrophages and is believed to play a major role in the development of chronic obstructive pulmonary disease (COPD). The catalytic domain of MMP-12 is unique among MMPs in that it is very highly active on numerous substrates including elastin. However, measuring MMP-12 activity in biological fluids has been hampered by the lack of highly selective substrates. We therefore synthesized four series of fluorogenic peptide substrates based on the sequences of MMP-12 cleavage sites in its known substrates. Human MMP-12 efficiently cleaved peptide substrates containing a Pro at P3 in the sequence Pro-X-X↓Leu but lacked selectivity towards these substrates compared to other MMPs, including MMP-2, MMP-7, MMP-9 and MMP-13. On the contrary, the substrate Abz-RNALAVERTAS-EDDnp derived from the CXCR5 chemokine was the most selective substrate for MMP-12 ever reported. All substrates were cleaved more efficiently by full-length MMP-12 than by its catalytic domain alone, indicating that the C-terminal hemopexin domain influences substrate binding and/or catalysis. Docking experiments revealed unexpected interactions between the peptide substrate Abz-RNALAVERTAS-EDDn and MMP-12 residues. Most of our substrates were poorly cleaved by murine MMP-12 suggesting that human and murine MMP-12 have different substrate specificities despite their structural similarity.

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14

Slupe, Andrew M., Ronald A. Merrill, and Stefan Strack. "Determinants for Substrate Specificity of Protein Phosphatase 2A." Enzyme Research 2011 (July 2, 2011): 1–8. http://dx.doi.org/10.4061/2011/398751.

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Protein phosphatase 2A- (PP2A-) catalyzed dephosphorylation of target substrate proteins is widespread and critical for cellular function. PP2A is predominantly found as a heterotrimeric complex of a catalytic subunit (C), a scaffolding subunit (A), and one member of 4 families of regulatory subunits (B). Substrate specificity of the holoenzyme complex is determined by the subcellular locale the complex is confined to, selective incorporation of the B subunit, interactions with endogenous inhibitory proteins, and specific intermolecular interactions between PP2A and target substrates. Here, we discuss recent studies that have advanced our understanding of the molecular determinants for PP2A substrate specificity.

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15

Sen, Liu, Dong Pei, Song Liu, and Xiao Hong Ma. "A Computational Model for Predicting ADAM17 Substrate Specificity." Advanced Materials Research 740 (August 2013): 525–29. http://dx.doi.org/10.4028/www.scientific.net/amr.740.525.

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Tumor necrosis factor-alpha converting enzyme (TACE) is a membrane-anchored protein that releases the soluble forms of many proteins by a process called ectodomain shedding. TACE has been considered as a potential target in a lot of diseases in autoimmune diseases and in cancers recently. In spite a lot of protein substrates have been found these years for TACE, the substrate selection of TACE is still not known. In this paper, a TACE-peptide complex was constructed, and used for the prediction of substrate sequences and cleavage sites. The result could be useful for understanding the substrate specificity of TACE, and designing better TACE inhibitors in future.

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16

Naegeli, Andreas, Gaëlle Michaud, Mario Schubert, Chia-Wei Lin, Christian Lizak, Tamis Darbre, Jean-Louis Reymond, and Markus Aebi. "Substrate Specificity of CytoplasmicN-Glycosyltransferase." Journal of Biological Chemistry 289, no. 35 (June 24, 2014): 24521–32. http://dx.doi.org/10.1074/jbc.m114.579326.

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17

Marsh, Ian R., and Mark Bradley. "Substrate Specificity of Trypanothione Reductase." European Journal of Biochemistry 243, no. 3 (February 1997): 690–94. http://dx.doi.org/10.1111/j.1432-1033.1997.00690.x.

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18

Beard, William A., David D. Shock, Vinod K. Batra, Lars C. Pedersen, and Samuel H. Wilson. "DNA Polymerase β Substrate Specificity." Journal of Biological Chemistry 284, no. 46 (September 15, 2009): 31680–89. http://dx.doi.org/10.1074/jbc.m109.029843.

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19

Hajela, K., and R. B. Sim. "Substrate specificity of MASP-1." Biochemical Society Transactions 30, no. 5 (October 1, 2002): A117. http://dx.doi.org/10.1042/bst030a117b.

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20

Bheda, Poonam, Hui Jing, Cynthia Wolberger, and Hening Lin. "The Substrate Specificity of Sirtuins." Annual Review of Biochemistry 85, no. 1 (June 2, 2016): 405–29. http://dx.doi.org/10.1146/annurev-biochem-060815-014537.

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21

Zong, Ning, Yoshi Kamiyama, and Tsuneo Yasui. "Substrate Specificity ofBacillusα-d-Xylosidase." Agricultural and Biological Chemistry 53, no. 8 (August 1989): 2129–39. http://dx.doi.org/10.1080/00021369.1989.10869623.

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22

Moore, S. P., M. Powers, and D. J. Garfinkel. "Substrate specificity of Ty1 integrase." Journal of virology 69, no. 8 (1995): 4683–92. http://dx.doi.org/10.1128/jvi.69.8.4683-4692.1995.

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23

Aloi, Sekotilani, Casey G. Davies, P. Andrew Karplus, Sigurd M. Wilbanks, and Guy N. L. Jameson. "Substrate Specificity in Thiol Dioxygenases." Biochemistry 58, no. 19 (May 2, 2019): 2398–407. http://dx.doi.org/10.1021/acs.biochem.9b00079.

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24

Nemmara, Venkatesh V., S. A. Adediran, Kinjal Dave, Colette Duez, and R. F. Pratt. "Dual Substrate Specificity ofBacillus subtilisPBP4a." Biochemistry 52, no. 15 (April 5, 2013): 2627–37. http://dx.doi.org/10.1021/bi400211q.

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25

Sentürker, Sema, Cécile Bauche, Jacques Laval, and Miral Dizdaroglu. "Substrate Specificity ofDeinococcus radioduransFpg Protein†." Biochemistry 38, no. 29 (July 1999): 9435–39. http://dx.doi.org/10.1021/bi990680m.

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26

Ruzindana-Umunyana, Angelique, Lise Imbeault, and Joseph M. Weber. "Substrate specificity of adenovirus protease." Virus Research 89, no. 1 (October 2002): 41–52. http://dx.doi.org/10.1016/s0168-1702(02)00111-9.

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27

Schendel, F. J., and J. Stubbe. "Substrate specificity of formylglycinamidine synthetase." Biochemistry 25, no. 8 (April 1986): 2256–64. http://dx.doi.org/10.1021/bi00356a061.

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28

Bulychev, Nikolai V., Chamakura V. Varaprasad, György Dormán, Jeffrey H. Miller, Moisés Eisenberg, Arthur P. Grollman, and Francis Johnson. "Substrate Specificity ofEscherichia coliMutY Protein†." Biochemistry 35, no. 40 (January 1996): 13147–56. http://dx.doi.org/10.1021/bi960694h.

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29

Langston, James, Alexander Blinkovsky, Tony Byun, Michael Terribilini, Darron Ransbarger, and Feng Xu. "Substrate specificity of Streptomyces transglutaminases." Applied Biochemistry and Biotechnology 136, no. 3 (March 2007): 291–308. http://dx.doi.org/10.1007/s12010-007-9027-5.

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30

McCoy, Elizabeth, M. Carmen Galan, and Sarah E. O’Connor. "Substrate specificity of strictosidine synthase." Bioorganic & Medicinal Chemistry Letters 16, no. 9 (May 2006): 2475–78. http://dx.doi.org/10.1016/j.bmcl.2006.01.098.

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31

Clary, Greg L., Cheu-Fen Tsai, and Robert W. Guynn. "Substrate specificity of choline kinase." Archives of Biochemistry and Biophysics 254, no. 1 (April 1987): 214–21. http://dx.doi.org/10.1016/0003-9861(87)90097-x.

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32

McLarin, Mark-Anthony, and Ivanhoe K. H. Leung. "Substrate specificity of polyphenol oxidase." Critical Reviews in Biochemistry and Molecular Biology 55, no. 3 (May 3, 2020): 274–308. http://dx.doi.org/10.1080/10409238.2020.1768209.

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33

Sebti, Said M., John C. Deleon, Ling-Tai Ma, Sidney M. Hecht, and John S. Lazo. "Substrate specificity of bleomycin hydrolase." Biochemical Pharmacology 38, no. 1 (January 1989): 141–47. http://dx.doi.org/10.1016/0006-2952(89)90160-3.

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34

Magnani, Roberta, Nihar R. Nayak, Mitra Mazarei, Lynnette M. A. Dirk, and Robert L. Houtz. "Polypeptide Substrate Specificity of PsLSMT." Journal of Biological Chemistry 282, no. 38 (July 17, 2007): 27857–64. http://dx.doi.org/10.1074/jbc.m702069200.

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Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P-3 to P+3) were engineered in the fusion protein. The catalytic efficiency (kcat/Km) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-π, where X is any residue, Lys is the methylation site, and π is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including γ-tocopherol methyltransferase (γ-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of γ-TMT from Perilla frutescens confirmed recognition and methylation of γ-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of α-tocopherol, the product of γ-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.

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35

Raven, Emma L., Latesh Lad, Katherine H. Sharp, Martin Mewies, and Peter C. E. Moody. "Defining substrate specificity and catalytic mechanism in ascorbate peroxidase." Biochemical Society Symposia 71 (March 1, 2004): 27–38. http://dx.doi.org/10.1042/bss0710027.

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Haem peroxidases catalyse the H2O2-dependent oxidation of a variety of, usually organic, substrates. Mechanistically, these enzymes are very well characterized: they share a common catalytic cycle that involves formation of a two-electron oxidized intermediate (Compound I) followed by reduction of Compound I by substrate. The substrate specificity is more diverse, however. Most peroxidases oxidize small organic substrates, but there are prominent exceptions to this and the structural features that control substrate specificity remain poorly defined. APX (ascorbate peroxidase) catalyses the H2O2-dependent oxidation of l-ascorbate and has properties that place it at the interface between the class I (e.g. cytochrome c peroxidase) and classical class III (e.g. horseradish peroxidase) peroxidase enzymes. We present a unified analysis of the catalytic and substrate-binding properties of APX, including the crystal structure of the APX-ascorbate complex. Our results provide new rationalization of the unusual functional features of the related cytochrome c peroxidase enzyme, which has been a benchmark for peroxidase-mediated catalysis for more than 20 years.

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36

Seo, Gil-Ju, Se-Eun Kim, Young-Man Lee, Jeong-Won Lee, Jae-Rin Lee, Myong-Joon Hahn, and Seong-Tae Kim. "Determination of substrate specificity and putative substrates of Chk2 kinase." Biochemical and Biophysical Research Communications 304, no. 2 (May 2003): 339–43. http://dx.doi.org/10.1016/s0006-291x(03)00589-8.

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37

Kobe, Boštjan, Thorsten Kampmann, Jade K. Forwood, Pawel Listwan, and Ross I. Brinkworth. "Substrate specificity of protein kinases and computational prediction of substrates." Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics 1754, no. 1-2 (December 2005): 200–209. http://dx.doi.org/10.1016/j.bbapap.2005.07.036.

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38

Raymond, Amy C., Bart L. Staker, and Alex B. Burgin. "Substrate Specificity of Tyrosyl-DNA Phosphodiesterase I (Tdp1)." Journal of Biological Chemistry 280, no. 23 (April 4, 2005): 22029–35. http://dx.doi.org/10.1074/jbc.m502148200.

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Tyrosyl-DNA phosphodiesterase I (Tdp1) hydrolyzes 3′-phosphotyrosyl bonds to generate 3′-phosphate DNA and tyrosine in vitro. Tdp1 is involved in the repair of DNA lesions created by topoisomerase I, although the in vivo substrate is not known. Here we study the kinetic and binding properties of human Tdp1 (hTdp1) to identify appropriate 3′-phosphotyrosyl DNA substrates. Genetic studies argue that Tdp1 is involved in double and single strand break repair pathways; however, x-ray crystal structures suggest that Tdp1 can only bind single strand DNA. Separate kinetic and binding experiments show that hTdp1 has a preference for single-stranded and blunt-ended duplex substrates over nicked and tailed duplex substrate conformations. Based on these results, we present a new model to explain Tdp1/DNA binding properties. These results suggest that Tdp1 only acts upon double strand breaks in vivo, and the roles of Tdp1 in yeast and mammalian cells are discussed.

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39

Yamawaki, Yuuki, Tomoki Yufu, and Tamaki Kato. "The Effect of a Peptide Substrate Containing an Unnatural Branched Amino Acid on Chymotrypsin Activity." Processes 9, no. 2 (January 28, 2021): 242. http://dx.doi.org/10.3390/pr9020242.

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7-Amino-4-methylcoumarin (AMC) is a low molecular weight fluorescent probe that can be attached to a peptide to enable the detection of specific proteases, such as chymotrypsin, expressed in certain diseases. Because this detection depends on the specificity of the protease toward the peptidyl AMC, the development of specific substrates is required. To investigate the specificity of chymotrypsin, peptidyl AMC compounds incorporating four different amino acid residues were prepared by liquid-phase synthesis. Two unnatural amino acids, 2-amino-4-ethylhexanoic acid (AEH) and cyclohexylalanine (Cha), were used to investigate the substrate specificity as these amino acids have structures different from natural amino acids. AEH was synthesized using diethyl acetamidemalonate as a starting material. The substrate containing Cha had high hydrophobicity and showed a high reaction velocity with chymotrypsin. Although the AEH substrate with a branched side chain had high hydrophobicity, it showed a low reaction velocity. The substrate containing the aromatic amino acid phenylalanine was less hydrophobic than the Cha and AEH substrates, but chymotrypsin showed the highest specificity for this compound. These results demonstrated that the substrate specificity of chymotrypsin is not only affected by the hydrophobicity and aromaticity, but also by the structural expanse of amino acid residues in the substrate.

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40

Knape, Matthias J., Maximilian Wallbott, Nicole C. G. Burghardt, Daniela Bertinetti, Jan Hornung, Sven H. Schmidt, Robin Lorenz, and Friedrich W. Herberg. "Molecular Basis for Ser/Thr Specificity in PKA Signaling." Cells 9, no. 6 (June 25, 2020): 1548. http://dx.doi.org/10.3390/cells9061548.

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cAMP-dependent protein kinase (PKA) is the major receptor of the second messenger cAMP and a prototype for Ser/Thr-specific protein kinases. Although PKA strongly prefers serine over threonine substrates, little is known about the molecular basis of this substrate specificity. We employ classical enzyme kinetics and a surface plasmon resonance (SPR)-based method to analyze each step of the kinase reaction. In the absence of divalent metal ions and nucleotides, PKA binds serine (PKS) and threonine (PKT) substrates, derived from the heat-stable protein kinase inhibitor (PKI), with similar affinities. However, in the presence of metal ions and adenine nucleotides, the Michaelis complex for PKT is unstable. PKA phosphorylates PKT with a higher turnover due to a faster dissociation of the product complex. Thus, threonine substrates are not necessarily poor substrates of PKA. Mutation of the DFG+1 phenylalanine to β-branched amino acids increases the catalytic efficiency of PKA for a threonine peptide substrate up to 200-fold. The PKA Cα mutant F187V forms a stable Michaelis complex with PKT and shows no preference for serine versus threonine substrates. Disease-associated mutations of the DFG+1 position in other protein kinases underline the importance of substrate specificity for keeping signaling pathways segregated and precisely regulated.

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Parent, Kristin N., and Carolyn M. Teschke. "GroEL/S substrate specificity based on substrate unfolding propensity." Cell Stress & Chaperones 12, no. 1 (2007): 20. http://dx.doi.org/10.1379/csc-219r.1.

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42

Uchida, S., and H. Endou. "Substrate specificity to maintain cellular ATP along the mouse nephron." American Journal of Physiology-Renal Physiology 255, no. 5 (November 1, 1988): F977—F983. http://dx.doi.org/10.1152/ajprenal.1988.255.5.f977.

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To evaluate substrate utilization along the mouse nephron, cellular ATP content was measured under various conditions by the luciferin-luciferase technique. Individual micro-dissected nephron segments were incubated in a modified Hanks' solution (pH 7.4) with or without each of the following substrates: D-glucose, DL-lactate, beta-hydroxybutyrate (HBA), and L-glutamine. ATP production from glucose was minimal in the early proximal tubule (S1), but was substantial in the late proximal tubule (S3). Glutamine and lactate were the preferred substrates in proximal tubules. In contrast, ATP production from glutamine was less than that from the other substrates in distal nephron segments, including medullary and cortical thick ascending limbs of Henle's loop (MTAL and CTAL), distal tubule including the connecting tubule (DT), and cortical and medullary collecting tubules (CCT and MCT). Lactate and HBA were preferred substrates for ATP maintenance in CTAL, MTAL, and DT. Glucose was the best substrate in CCT. In addition, the specific contribution of anaerobic metabolism to maintaining cellular ATP was low in MTAL and CTAL. On the other hand, the glycolytic capacity of CCT and MCT was high. The above results demonstrate the substrate requirements for maintaining cellular ATP content within specific nephron segments.

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Goto, Yoshikuni, Hiroe Tanji, Akira Hattori, and Masafumi Tsujimoto. "Glutamine-181 is crucial in the enzymatic activity and substrate specificity of human endoplasmic-reticulum aminopeptidase-1." Biochemical Journal 416, no. 1 (October 28, 2008): 109–16. http://dx.doi.org/10.1042/bj20080965.

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ERAP-1 (endoplasmic-reticulum aminopeptidase-1) is a multifunctional enzyme with roles in the regulation of blood pressure, angiogenesis and the presentation of antigens to MHC class I molecules. Whereas the enzyme shows restricted specificity toward synthetic substrates, its substrate specificity toward natural peptides is rather broad. Because of the pathophysiological significance of ERAP-1, it is important to elucidate the molecular basis of its enzymatic action. In the present study we used site-directed mutagenesis to identify residues affecting the substrate specificity of human ERAP-1 and identified Gln181 as important for enzymatic activity and substrate specificity. Replacement of Gln181 by aspartic acid resulted in a significant change in substrate specificity, with Q181D ERAP-1 showing a preference for basic amino acids. In addition, Q181D ERAP-1 cleaved natural peptides possessing a basic amino acid at the N-terminal end more efficiently than did the wild-type enzyme, whereas its cleavage of peptides with a non-basic amino acid was significantly reduced. Another mutant enzyme, Q181E, also revealed some preference for peptides with a basic N-terminal amino acid, although it had little hydrolytic activity toward the synthetic peptides tested. Other mutant enzymes, including Q181N and Q181A ERAP-1s, revealed little enzymatic activity toward synthetic or peptide substrates. These results indicate that Gln181 is critical for the enzymatic activity and substrate specificity of ERAP-1.

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44

Oizumi, J., and K. Hayakawa. "Lipoamidase is a multiple hydrolase." Biochemical Journal 271, no. 1 (October 1, 1990): 45–49. http://dx.doi.org/10.1042/bj2710045.

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The substrate specificity of lipoamidase, purified from the pig brain membrane with lipoyl 4-aminobenzoate (LPAB) as a substrate, was extensively studied. This single polypeptide was found to hydrolyse the bonding between amide, ester and peptide compounds. However, stringent structural requirements were found in the substrates, e.g. LPAB was hydrolysed, whereas biotinyl 4-aminobenzoate was not, as stated in our previous paper [Oizmui & Hayakawa (1990) Biochem. J. 266, 427-434]. The enzyme specifically recognized the whole molecular structure of the substrate, whereas it loosely recognized the bond structure of the substrate; e.g. the dipeptide Asp-Phe was not hydrolysed, whereas the methyl ester of Asp-Phe (aspartame) was. The exopeptidase activity was demonstrated by lipoamidase; however, longer peptides than the hexamer seemed not to be substrates. Lipoyl esters, which were electrically neutral, exhibited higher specificity with longer acyl groups. Molecular mass and molecular hydrophobicity (hydropathy) seemed to determine the substrate specificity. Lipoyl-lysine, acetylcholine and oligopeptides were hydrolysed at similar Km values; however, acetylcholine was hydrolysed at a velocity 100 times higher. Although many similar specificities were found between electric eel acetylcholinesterase and lipoamidase, distinctly different specificity was demonstrated with lipoyl compounds. The role of lipoamidase, which resides on the brain membrane and possesses higher specificity for hydrophobic molecules, remains to be elucidated.

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BERRIN, Jean-Guy, Mirjam CZJZEK, Paul A. KROON, W. Russell MCLAUCHLAN, Antoine PUIGSERVER, Gary WILLIAMSON, and Nathalie JUGE. "Substrate (aglycone) specificity of human cytosolic beta-glucosidase." Biochemical Journal 373, no. 1 (July 1, 2003): 41–48. http://dx.doi.org/10.1042/bj20021876.

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Human cytosolic β-glucosidase (hCBG) is a xenobiotic-metabolizing enzyme that hydrolyses certain flavonoid glucosides, with specificity depending on the aglycone moiety, the type of sugar and the linkage between them. Based upon the X-ray structure of Zea mays β-glucosidase, we generated a three-dimensional model of hCBG by homology modelling. The enzyme exhibited the (β/α)8-barrel fold characteristic of family 1 β-glucosidases, with structural differences being confined mainly to loop regions. Based on the substrate specificity of the human enzymes, sequence alignment of family 1 enzymes and analysis of the hCBG structural model, we selected and mutated putative substrate (aglycone) binding site residues. Four single mutants (Val168→Tyr, Phe225→Ser, Tyr308→Ala and Tyr308→Phe) were expressed in Pichia pastoris, purified and characterized. All mutant proteins showed a decrease in activity towards a broad range of substrates. The Val168→Tyr mutation did not affect Km on p-nitrophenyl (pNP)-glycosides, but increased Km 5-fold on flavonoid glucosides, providing the first biochemical evidence supporting a role for this residue in aglycone-binding of the substrate, a finding consistent with our three-dimensional model. The Phe225→Ser and Tyr308→Ala mutations, and, to a lesser degree, the Tyr308→Phe mutation, resulted in a drastic decrease in specific activities towards all substrates tested, indicating an important role of those residues in catalysis. Taken together with the three-dimensional model, these mutation studies identified the amino-acid residues in the aglycone-binding subsite of hCBG that are essential for flavonoid glucoside binding and catalysis.

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Fan, Anwen, Ziyao Wang, Haojie Qu, Yao Nie, and Yan Xu. "Semi-Rational Design of Proteus mirabilis l-Amino Acid Deaminase for Expanding Its Substrate Specificity in α-Keto Acid Synthesis from l-Amino Acids." Catalysts 12, no. 2 (January 29, 2022): 175. http://dx.doi.org/10.3390/catal12020175.

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l-amino acid deaminases (LAADs) are flavoenzymes that catalyze the stereospecific oxidative deamination of l-amino acids into α-keto acids, which are widely used in the pharmaceutical, food, chemical, and cosmetic industries. However, the substrate specificity of available LAADs is limited, and most substrates are concentrated on several bulky or basic l-amino acids. In this study, we employed a LAAD from Proteus mirabilis (PmiLAAD) and broadened its substrate specificity using a semi-rational design strategy. Molecular docking and alanine scanning identified F96, Q278, and E417 as key residues around the substrate-binding pocket of PmiLAAD. Site-directed saturation mutagenesis identified E417 as the key site for substrate specificity expansion. Expansion of the substrate channel with mutations of E417 (E417L, E417A) improved activity toward the bulky substrate l-Trp, and mutation of E417 to basic amino acids (E417K, E417H, E417R) enhanced the universal activity toward various l-amino acid substrates. The variant PmiLAADE417K showed remarkable catalytic activity improvement on seven substrates (l-Ala, l-Asp, l-Ile, l-Leu, l-Phe, l-Trp, and l-Val). The catalytic efficiency improvement obtained by E417 mutation may be attributed to the expansion of the entrance channel and its electrostatic interactions. These PmiLAAD variants with a broadened substrate spectrum can extend the application potential of LAADs.

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Garcia-Pardo, Javier, Sebastian Tanco, Maria C. Garcia-Guerrero, Sayani Dasgupta, Francesc Xavier Avilés, Julia Lorenzo, and Lloyd D. Fricker. "Substrate Specificity and Structural Modeling of Human Carboxypeptidase Z: A Unique Protease with a Frizzled-Like Domain." International Journal of Molecular Sciences 21, no. 22 (November 18, 2020): 8687. http://dx.doi.org/10.3390/ijms21228687.

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Metallocarboxypeptidase Z (CPZ) is a secreted enzyme that is distinguished from all other members of the M14 metallocarboxypeptidase family by the presence of an N-terminal cysteine-rich Frizzled-like (Fz) domain that binds Wnt proteins. Here, we present a comprehensive analysis of the enzymatic properties and substrate specificity of human CPZ. To investigate the enzymatic properties, we employed dansylated peptide substrates. For substrate specificity profiling, we generated two different large peptide libraries and employed isotopic labeling and quantitative mass spectrometry to study the substrate preference of this enzyme. Our findings revealed that CPZ has a strict requirement for substrates with C-terminal Arg or Lys at the P1′ position. For the P1 position, CPZ was found to display specificity towards substrates with basic, small hydrophobic, or polar uncharged side chains. Deletion of the Fz domain did not affect CPZ activity as a carboxypeptidase. Finally, we modeled the structure of the Fz and catalytic domains of CPZ. Taken together, these studies provide the molecular elucidation of substrate recognition and specificity of the CPZ catalytic domain, as well as important insights into how the Fz domain binds Wnt proteins to modulate their functions.

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Chen, Xiaobo, Jiayue Chen, Bing Yan, Wei Zhang, Luke W. Guddat, Xiang Liu, and Zihe Rao. "Structural basis for the broad substrate specificity of two acyl-CoA dehydrogenases FadE5 from mycobacteria." Proceedings of the National Academy of Sciences 117, no. 28 (June 29, 2020): 16324–32. http://dx.doi.org/10.1073/pnas.2002835117.

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FadE, an acyl-CoA dehydrogenase, introduces unsaturation to carbon chains in lipid metabolism pathways. Here, we report that FadE5 fromMycobacterium tuberculosis(MtbFadE5) andMycobacterium smegmatis(MsFadE5) play roles in drug resistance and exhibit broad specificity for linear acyl-CoA substrates but have a preference for those with long carbon chains. Here, the structures ofMsFadE5 andMtbFadE5, in the presence and absence of substrates, have been determined. These reveal the molecular basis for the broad substrate specificity of these enzymes. FadE5 interacts with the CoA region of the substrate through a large number of hydrogen bonds and an unusual π–π stacking interaction, allowing these enzymes to accept both short- and long-chain substrates. Residues in the substrate binding cavity reorient their side chains to accommodate substrates of various lengths. Longer carbon-chain substrates make more numerous hydrophobic interactions with the enzyme compared with the shorter-chain substrates, resulting in a preference for this type of substrate.

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Dauber, Deborah S., Rainer Ziermann, Neil Parkin, Dustin J. Maly, Sami Mahrus, Jennifer L. Harris, Jon A. Ellman, Christos Petropoulos, and Charles S. Craik. "Altered Substrate Specificity of Drug-Resistant Human Immunodeficiency Virus Type 1 Protease." Journal of Virology 76, no. 3 (February 1, 2002): 1359–68. http://dx.doi.org/10.1128/jvi.76.3.1359-1368.2002.

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ABSTRACT Resistance to human immunodeficiency virus type 1 protease (HIV PR) inhibitors results primarily from the selection of multiple mutations in the protease region. Because many of these mutations are selected for the ability to decrease inhibitor binding in the active site, they also affect substrate binding and potentially substrate specificity. This work investigates the substrate specificity of a panel of clinically derived protease inhibitor-resistant HIV PR variants. To compare protease specificity, we have used positional-scanning, synthetic combinatorial peptide libraries as well as a select number of individual substrates. The subsite preferences of wild-type HIV PR determined by using the substrate libraries are consistent with prior reports, validating the use of these libraries to compare specificity among a panel of HIV PR variants. Five out of seven protease variants demonstrated subtle differences in specificity that may have significant impacts on their abilities to function in viral maturation. Of these, four variants demonstrated up to fourfold changes in the preference for valine relative to alanine at position P2 when tested on individual peptide substrates. This change correlated with a common mutation in the viral NC/p1 cleavage site. These mutations may represent a mechanism by which severely compromised, drug-resistant viral strains can increase fitness levels. Understanding the altered substrate specificity of drug-resistant HIV PR should be valuable in the design of future generations of protease inhibitors as well as in elucidating the molecular basis of regulation of proteolysis in HIV.

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50

Matoušek, Jaroslav. "The substrate specificity of pollen extracellular nuclease." Biochemistry and Cell Biology 64, no. 9 (September 1, 1986): 891–97. http://dx.doi.org/10.1139/o86-119.

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A detailed study of substrate specificity of tobacco pollen extracellular nuclease showed that the nuclease degraded, in order of decreasing rate, poly(U), RNA, heat-denatured DNA (ssDNA), poly(dA), poly(dA)∙poly(dT) duplex, and native or undenatured DNA (dsDNA). Poly(dC) was cleaved at a very low rate, while the poly(dC)∙poly(dG) duplex was degraded only at a negligible rate (0.8% of that with dsDNA). Poly(dG) was not detectably cleaved. The nuclease exhibited lower activity towards AG and GC copolymers than with AC and AU copolymers. Preference of the nuclease for ssDNA over dsDNA strongly depended on the ionic strength. The nuclease exhibited no preference for alkylated native DNA versus native DNA and degraded apurinated DNA at a lower rate. It also cleaved ultraviolet-irradiated DNA, but the cleavage did not lead to the specific excision of pyrimidine photodimers. Two molecular forms of the tobacco pollen nuclease isolated from polyacrylamide gels showed the same substrate specificity. The extracellular nucleases of five other pollen species examined also cleaved the following substrates in order of decreasing rate: poly(U) > ssDNA > poly(dA) > dsDNA. In each case poly(dG) was resistant to cleavage.

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