Enzyme active-site residues

The reaction path of thiamine-dependent catalysis is essentially unchanged in the presence of an apoenzyme, except that the enzyme active site residues increase reaction rates and yields and influence the substrate and product specificity. The X-ray crystal structures of TDP-dependent enzymes have clarified this view and permitted an understanding of the roles of the individual amino acids of the active site in activating and controlling the thiazolium reactivity [36-40]. 

The precise knowledge of major physical components involved in such interactions is essential to understand biological function of enzyme active site residues and derivation of simplified methods representing environmental effects in chemical reactions. Therefore we attempted to analyze the nature of intermolecular forces between reactants at various stages of enzyme reaction and different forms of catalytic residues. 

Specificity for a particular charged substrate can be engineered into an enzyme by replacement of residues within the enzyme-active site to achieve electrostatic complementarity between the enzyme and substrate (75). Protein engineering, when coupled with detailed stmctural information, is a powerful technique that can be used to alter the catalytic activity of an enzyme in a predictable fashion. 

In free CDK2 the active site cleft is blocked by the T-loop and Thr 160 is buried (Figure 6.20a). Substrates cannot bind and Thr 160 cannot be phosphorylated consequently free CDK2 is inactive. The conformational changes induced by cyclin A binding not only expose the active site cleft so that ATP and protein substrates can bind but also rearrange essential active site residues to make the enzyme catalytically competent (Figure 6.20b). In addition Thr... 

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with C. Acid hydrolysis of the inactivated enzyme liberates a novel C-labeled amino acid, N -dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the C-labeled dihydroxy-acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of C labeling in a case such as this facilitates the separation and identification of the telltale amino acid. 

A different mechanism operates in the wheat germ enzyme. 2,3-Bisphosphoglycerate is not a cofactor. Instead, the enzyme carries out intra-molecular phosphoryl group transfer (Figure 19.25). The C-3 phosphate is transferred to an active-site residue and then to the C-2 position of the original substrate molecule to form the product, 2-phosphoglycerate. 

Hen egg-white lysozyme catalyzes the hydrolysis of various oligosaccharides, especially those of bacterial cell walls. The elucidation of the X-ray structure of this enzyme by David Phillips and co-workers (Ref. 1) provided the first glimpse of the structure of an enzyme-active site. The determination of the structure of this enzyme with trisaccharide competitive inhibitors and biochemical studies led to a detailed model for lysozyme and its hexa N-acetyl glucoseamine (hexa-NAG) substrate (Fig. 6.1). These studies identified the C-O bond between the D and E residues of the substrate as the bond which is being specifically cleaved by the enzyme and located the residues Glu 37 and Asp 52 as the major catalytic residues. The initial structural studies led to various proposals of how catalysis might take place. Here we consider these proposals and show how to examine their validity by computer modeling approaches. 

Similar reaction mechanisms, involving general base and metal ion catalysis, in conjunction with an OH nucleophilic attack, have been proposed for thermolysin (Ref. 12) and carboxypeptidase A (Refs. 12 and 13). Both these enzymes use Zn2+ as their catalytic metal and they also have additional positively charged active site residues (His 231 in thermolysin and... 

Fructose-2,6-bisphosphatase, a regulatory enzyme of gluconeogenesis (Chapter 19), catalyzes the hydrolytic release of the phosphate on carbon 2 of fructose 2,6-bisphosphate. Figure 7-8 illustrates the roles of seven active site residues. Catalysis involves a catalytic triad of one Glu and two His residues and a covalent phos-phohistidyl intermediate. 

Selenocysteine, an essential active site residue in several mammahan enzymes, arises by co-translational insertion of a previously modified tRNA. 

However, diffusion of the reactive QM out of the enzyme active site is a major concern. For instance, a 2-acyloxy-5-nitrobenzylchloride does not modify any nucleophilic residue located within the enzyme active site but becomes attached to a tryptophan residue proximal to the active site of chymotrypsin or papain.23,24 The lack of inactivation could also be due to other factors the unmasked QM being poorly electrophilic, active site residues not being nucleophilic enough, or the covalent adduct being unstable. Cyclized acyloxybenzyl molecules of type a could well overcome the diffusion problem. They will retain both the electrophilic hydroxybenzyl species b, and then the tethered QM, in the active site throughout the lifetime of the acyl-enzyme (Scheme 11.1). This reasoning led us to synthesize functionalized... 

The studies described above show that a quinone methide or its aza-analogue quinonimine methide incorporated as a latent electrophilic species into a cyclic lactone or lactam precursor can modify a second nucleophilic residue within the enzyme active site after formation of the acyl-enzyme. Very efficient suicide... 

One reason for the relatively large RMS deviations, compared to the active sites of MMO and RNR, is that the active-site residues are not coordinated to the selenium (see Figure 2-8). The lack of a structural anchor leads to a relatively unstable active-site geometry. An alternative formulation is that the presence of a metal center with strong ligand interactions is one reason the active-site model works comparatively well for many metal enzymes. 

Before our work [39], only one catalytic mechanism for zinc dependent HDACs has been proposed in the literature, which was originated from the crystallographic study of HDLP [47], a histone-deacetylase-like protein that is widely used as a model for class-I HDACs. In the enzyme active site, the catalytic metal zinc is penta-coordinated by two asp residues, one histidine residues as well as the inhibitor [47], Based on their crystal structures, Finnin et al. [47] postulated a catalytic mechanism for HDACs in which the first reaction step is analogous to the hydroxide mechanism for zinc proteases zinc-bound water is a nucleophile and Zn2+ is five-fold coordinated during the reaction process. However, recent experimental studies by Kapustin et al. suggested that the transition state of HDACs may not be analogous to zinc-proteases [48], which cast some doubts on this mechanism. 

The fitting was not successful for the peptide-aldehyde inhibited form of the SGPA enzyme underlining the alignment difference between the active site residues His57 and Asp 102 between the two forms of this enzyme (cf. Ref. 118). 

Coordinates of the SGPA active site residue and of the two water molecules W184 and W210 are from a further refinement of the published (1.8 A resolution) structure (Ref. 118b) with a resolution extending beyond 1.7 A for the native enzyme James, M. N. G, Sielecki, A. R. Private communications (1983, 1984)... 

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