Substrate-binding residues

Lappalainen, P., Watmough, N. J., Greenwood, C., and Saraste, M., 1995, Electron transfer between cytochrome c and the isolated Cu, domain identification of substrate-binding residues in cytochrome c oxidase. Biochemistry, 34 582495830. 

Nakajima K, Kato H, Oda J, Yamada Y, Hashimoto T. Site directed mutagenesis of putative substrate binding residues reveals a mechanism controlling different substrate specificities of two tropinone reductases. J. Biol. Chem. 1999 274 16563-16568. Yamashita A, Kato H, Wakatsuki S, Tomizaki T, Nakatsu T, Nakajima K, Hashimoto T, Yamada Y, Oda J. Structure of tropinone reductase-II with NADP + and pseudotropine at 1.9A resolution implication for stereospecific substrate binding and catalysis. Biochemistry 1999 38 7630-7637. 

Prior to the determination of the X-ray crystal structure of HIV-1 protease, a model of the enzyme was derived from the homologous RS V protease [56]. Within a short amount of time, the first crystal structures of the native HIV-1 protease were solved [57,58], followed by numerous enzyme/inhibitor crystal complexes [59]. These experimental results not only confirmed the accuracy of the model with regard to the substrate binding residues, but provided... 

The proposed models were consistent with site-directed mutagenesis results and later verified by the crystal structure (Fig. 27.4). In particular, the location of the active site and substrate binding residues were accurately predicted. This story demonstrates that modelling of unknown structures based on related proteins can give accurate results. In this case the two original models provided sufficient insight into the structure and function... 

Nakajima K, Kato H, Oda J et al (1999) Site-directed mutagenesis of putative substrate-binding residues reveals a mechanism controlling the different stereospecificities of two tropinone reductases. J Biol Chem 274 16563-16568... 

P-Lactam antibiotics exert their antibacterial effects via acylation of a serine residue at the active site of the bacterial transpeptidases. Critical to this mechanism of action is a reactive P-lactam ring having a proximate anionic charge that is necessary for positioning the ring within the substrate binding cleft (24). 

Figure 11.10 Topological diagram of the two domains of chymotrypsin, illustrating that the essential active-site residues are part of the same two loop regions (3-4 and 5-6, red) of the two domains. These residues form the catalytic triad, the oxyanion hole (green), and the substrate binding regions (yellow and blue) including essential residues in the specificity pocket.

This idea also helps to explain some of the mystery surrounding the enormous catalytic power of enzymes In enzyme catalysis, precise orientation of catalytic residues comprising the active site is necessary for the reaction to occur substrate binding induces this precise orientation by the changes it causes in the protein s conformation. 

Destabilization of the ES complex can involve structural strain, desolvation, or electrostatic effects. Destabilization by strain or distortion is usually just a consequence of the fact (noted previously) that the enzyme is designed to bind the transition state more strongly than the substrate. When the substrate binds, the imperfect nature of the fit results in distortion or strain in the substrate, the enzyme, or both. This means that the amino acid residues that make up the active site are oriented to coordinate the transition-state structure precisely, but will interact with the substrate or product less effectively. 

Cleavage occur s at the scissile bond. Residues in the substrate towards the N-terminus are numbered PI, P2, P3, etc, whereas residues towards the C-terminus are numbered PI, P2, P3 etc. Cleavage occurs between PI and P1. For a peptidase with limited specificity, only the residue in PI or PI is important for specificity. A peptidase with an extended substrate binding site will have a preference for residues in other positions. For example cathepsin L prefers substrates with phenylalanine in P2 and arginine in PI. However, this is a preference only, and cathepsin L cleaves substrates after other amino acids. Caspase-3 has a preference for Asp in both P4 and PI, but it is unusual for substrate specificity to extend much further from the scissile bond. The peptidase with the most extended substrate specificity may be mitochondrial intermediate peptidase that removes an octopeptide targeting signal from the N-terminus of cytoplasmically synthesized proteins that are destined for import into the mitochondrial lumen. 

Site-directed mutagenesis, used to change residues suspected of being important in catalysis or substrate binding, provides insights into the mechanisms of enzyme action. 

In the aligned primary structures of class I decarboxylases, the conserved amino acid residues are scattered over their primary structures. There have been few reports to identify the amino acid residues essential for catalytic activity or substrate binding. Huang et al. reported the E-X-P motif in the alignment analysis for 4-hydroxybenzoate decarboxylase of C. hydroxybenzoicum and its homologous unidentified proteins. The E-X-P motif is also conserved in pyrrole-2-carboxylate decarboxylase and indole-3-carboxylate decarboxylase (unpublished data). However, the corresponding motif sequence is not observed in the primary structures of 3,4-dihydroxybenzoate decarboxylase of E. cloacae P241. ... 

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