Substrate recognition sites

Fig. 22.3 Cytochrome P450 monooxygenases and nicotine metabolism. An alignment of the amino acid sequences of the enzymes 2A6 and 2D6. Occurrences of the same amino acid residue at the same position are shown in black. Putative substrate recognition sites (SRS1—...

Gotoh O. Substrate recognition sites in cytochrome P450 family 2 (CYP2) proteins inferred from comparative analyses of amino acid and coding nucleotide sequences. J Biol Chem 1992 267(1) 83-90. 

Domanski, T. L., He, Y. A., Khan, K. K., Roussel, F., Wang, Q., and Halpert, J. R. (2001) Phenylalanine and tryptophan scanning mutagenesis of CYP3A4 substrate recognition site residues and effect on substrate oxidation and coop-erativity. Biochemistry 40, 10,150-10,160. 

Carboxypeptidase A"" (CPA, EC 3.4.17.1) is a proteolytic enzyme that cleaves C-terminal amino acid residues with hydrophobic side chains selectively. Several X-ray structures are available" The active site of CPA consists of a hydrophobic pocket (primary substrate recognition site) that is primarily responsible for the substrate specificity, a guanidinium moiety of Argl45 that forms hydrogen bonds to the carboxylate of the substrate, and Glu270, whose carboxylate plays a critical role, functioning either as a nucleophile to attack the scissUe carboxamide carbonyl carbon of the substrate or as a base to activate the zinc-bound water molecule, which in turn attacks the scissile peptide bond ". However, semiempirical calculations had shown that the direct attack of... 

The direct thrombin inhibitors (DTIs) exert their anticoagulant effect by directly binding to the active site of thrombin, thereby inhibiting thrombin s downstream effects. This is in contrast to indirect thrombin inhibitors such as heparin and LMWH (see above), which act through antithrombin. Hirudin and bivalirudin are bivalent DTIs in that they bind at both the catalytic or active site of thrombin as well as at a substrate recognition site. Argatroban and melagatran are small molecules that bind only at the thrombin active site. 

P450 Analogues with Substrate Recognition Sites. 77... 

Fig. 27. An active site analogue of cytochrome P450 with substrate recognition sites and a thiophenolate ligand coordinating to the iron...

Fig. 28. Preparation of iron porphyrins with different chiral substrate recognition sites...

The new aldolase differs from all other existing ones with respect to the location of its active site in relation to its secondary structure and still displays enantiofacial discrimination during aldol addition. Modification of substrate specificity is achieved by altering the position of the active site lysine from one /3-strand to a neighboring strand rather than by modification of the substrate recognition site. Determination of the 3D crystal structure of the wild type and the double mutant demonstrated how catalytic competency is maintained despite spatial reorganization of the active site with respect to substrate. It is possible to perturb the active site residues themselves as well as surrounding loops to alter specificity. 

Domanski TL, Liu J, Harlow GR, Halpert JR (1998) Analysis of four residues within substrate recognition site 4 of human cytochrome P450 3A4 role in steroid hydroxylase activity and a-napthoflavone stimulation. Arch Biochem Biophys 350, 223-232. 

Harlow GR, Halpert JR (1997) Alanine-scanning mutagenesis of a putative substrate recognition site in human cytochrome P4503A4. J Biol Chem 272, 5396-5402. 

He YA, He YQ, Szklarz GD, Halpert JR (1997) Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis. Biochemistry 36, 8831-8839. 

A schematic of the structure of the thrombin molecule is presented in Figure 2. The important binding sites on the surface of the thrombin molecule include the catalytic site and two exosites (anionic and apolar) or substrate recognition sites. Whereas the catalytic site is responsible for the serine protease activity, the separate substrate recognition sites are involved in the binding of heparin, fibrinogen, and thrombomodulin (6). These sites serve as targets for the direct thrombin inhibitors,... 

A substrate recognition site that also functions as the binding site for the AT-heparin complex-AT complex (exosite I) and... 

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