The specificity of chymotrypsin

Section: Disorder-to-order Transition Of the Loop Is Confirmed By Limmentioning

confidence: 73%

Cross-talk between Thiamin Diphosphate Binding and Phosphorylation Loop Conformation in Human Branched-chain α-Keto Acid Decarboxylase/Dehydrogenase

Li¹,

Wynn

Machius³

et al. 2004

“…the S and SЈ subsites function independently. For example, simple additive behavior is seen with substrate hydrolysis by trypsin (16), chymotrypsin (17), tissue-type plasminogen activator (16), and subtilisin (18). Nonadditive interactions, i.e.…”

Section: Methodsmentioning

confidence: 99%

Distinct Multisite Synergistic Interactions Determine Substrate Specificities of Human Chymase and Rat Chymase-1 for Angiotensin II Formation and Degradation

Sanker

Chandrasekharan

Wilk

et al. 1997

Human chymase and rat chymase-1 are mast cell serine proteases involved in angiotensin II (Ang II) formation and degradation, respectively. Previous studies indicate that both these enzymes have similar P 1 and P 2 preferences, which are the major determinants of specificity. Surprisingly, despite the occurrence of optimal P 2 and P 1 residues at the Phe 82 . The overall effect of this P 1 Ile interaction on catalytic efficiency, however, is influenced by the structure of the acyl group and that of the other leaving group residues. For human chymase, the P 1 Ile interaction is not productive. Thus, specificity for Ang II formation versus Ang II degradation by these chymases is produced through synergistic interactions between acyl or leaving group residues as well as between the acyl and leaving groups. These observations indicate that nonadditive interactions between the extended substrate binding site of human chymase or rat chymase-1 and the substrate are best explained if the entire binding site is taken as an entity rather than as a collection of distinct subsites. Chymases1 are a family of mast cell serine proteinases involved in such diverse functions as inflammation (1), parasite expulsion (2), and peptide hormone processing (3-5). These serine proteinases are synthesized as inactive precursors but are stored in secretory granules as active enzymes (6). Recent phylogenetic evidence indicates that mammalian chymases occur as two distinct isoenzyme groups, ␣ and ␤ (7). ␣-Chymases include human chymase, dog chymase, mouse chymase-5, rat chymase-3, and gerbil chymase-2 (7, 8). ␤-Chymases include rat chymase-1 and -2, mouse chymase-1, -2, -4, and -L, and gerbil chymase-1 (7,8 . Early comparative studies on chymase specificities by Powers et al. (11) using peptide 4-nitroanilide substrates indicated that the S 1 to S 4 subsites 3 of human chymase and rat chymase-1 are similar. In both human chymase and rat chymase-1, the key features for optimal acyl group interactions are a P 1 hydrophobic aromatic residue, a P 2 hydrophobic residue or Pro, and a P 3 hydrophobic residue. S 4 subsite interactions are less restrictive. Thus, these studies could not explain why human chymase is an Ang II-forming enzyme and rat chymase-1 is an angiotensinase (3, 5) and suggested to us that enzyme-substrate interactions other than those occurring at the S 1 to S 4 subsites of these enzymes could be important for determining specificity. In a previous paper we explored the S 1 subsite as well as the SЈ 1 to SЈ 2 subsites of human chymase using decapeptide Ang I analogs. We showed that a P 1 hydrophobic aromatic residue was necessary but that several non-* This work was supported by National Institutes of Health Grant HL44201. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.‡ To whom correspondence should be addressed: Dept. of Molecular Cardiology, FF30,...

“…The pocket has three walls formed by residues 189 -195, 214 -220, and 225-228 (chymotrypsinogen numbering has been used for all SPs or SP domains throughout this paper) (3). The presence at the bottom of the pocket of Asp 189 endows trypsin with preference for positively charged Arg and Lys residues (4,5), whereas in chymotrypsin the specificity for bulky aromatics is largely determined by Ser 189 (6). Residues at position 216 and 226 also contribute to substrate specificity (7).…”

mentioning

confidence: 99%

Mutational Analysis of the Primary Substrate Specificity Pocket of Complement Factor B

Xu¹,

Circolo²,

Hua³

et al. 2000

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S 1 pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P 1 -Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp 226 is within the S 1 pocket, whereas Asp 187 is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp 187 caused moderate (50 -60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn 189 resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp 226 completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P 1 -Arg containing thioester by selected mutants confirmed that residue Asp 226 is a primary structural determinant for P 1 -Arg binding and catalysis.Complement is a major effector system of host defense. Activation of complement leads to the generation of protein fragments and protein-protein complexes that mediate acute inflammatory responses, phagocytosis and killing of pathogens, and regulation of adaptive immune responses. Activation-associated production of biologically active protein fragments is catalyzed by a group of eight atypical complement serine proteases (SPs) 1 of the chymotrypsin superfamily (1). Understanding the structural basis for the highly restricted proteolytic activity of these SPs is an important first step toward pharmacologic control of complement activation (2). Members of the chymotrypsin family have very similar three-dimensional structures but distinct substrate specificities. To a great extent specificity is determined by the side chains of the amino acid residues that line up the primary substrate specificity pocket (S 1 site). The pocket has three walls formed by residues 189 -195, 214 -220, and 225-228 (chymotrypsinogen numbering has been used for all SPs or SP domains throughout this paper) (3). The presence at the bottom of the pocket of Asp 189 endows trypsin with preference for positively charged Arg and Lys residues (4, 5), whereas in chymotrypsin the specificity for bulky aromatics is largely determined by Ser 189 (6). Residues at position 216 and 226 also contribute to substrate specificity (7). All complement SPs exhibit trypsinlike specificity for positively charged Arg residues and all have an Asp at position 189, except for factor B and C2 (Fig. 1).Factor B and C2 are structurally similar modular proteins that play a central role in complement activation by providing the catalytic subunits of...