Wild type enzymes

Model building also predicts that the Ala 216 mutant would displace a water molecule at the bottom of the specificity pocket that in the wild type enzyme binds to the NH3 group of the substrate Lys side chain (Figure 11.12). The extra CH3 group of this mutant is not expected to disturb the binding of the Arg side chain. One would therefore expect that the Km for Lys... 

The substrates used were D-Val-Leu-Arg-amino fluorocoumarin (Arg) and D-Val-Leu-Lys-amino fluorocoumarin (Lys). For clarity the Km and kcat values have been normalized to those of the wild-type enzyme for the Arg substrate. 

Asp 189 at the bottom of the substrate specificity pocket interacts with Lys and Arg side chains of the substrate, and this is the basis for the preferred cleavage sites of trypsin (see Figures 11.11 and 11.12). It is almost trivial to infer, from these observations, that a replacement of Asp 189 with Lys would produce a mutant that would prefer to cleave substrates adjacent to negatively charged residues, especially Asp. On a computer display, similar Asp-Lys interactions between enzyme and substrate can be modeled within the substrate specificity pocket but reversed compared with the wild-type enzyme. 

By changing Ser 221 in subtilisin to Ala the reaction rate (both kcat and kcat/Km) is reduced by a factor of about 10 compared with the wild-type enzyme. The Km value and, by inference, the initial binding of substrate are essentially unchanged. This mutation prevents formation of the covalent bond with the substrate and therefore abolishes the reaction mechanism outlined in Figure 11.5. When the Ser 221 to Ala mutant is further mutated by changes of His 64 to Ala or Asp 32 to Ala or both, as expected there is no effect on the catalytic reaction rate, since the reaction mechanism that involves the catalytic triad is no longer in operation. However, the enzyme still has an appreciable catalytic effect peptide hydrolysis is still about 10 -10 times the nonenzymatic rate. Whatever the reaction mechanism... 

Enzyme preparations from liver or microbial sources were reported to show rather high substrate specificity [76] for the natural phosphorylated acceptor d-(18) but, at much reduced reaction rates, offer a rather broad substrate tolerance for polar, short-chain aldehydes [77-79]. Simple aliphatic or aromatic aldehydes are not converted. Therefore, the aldolase from Escherichia coli has been mutated for improved acceptance of nonphosphorylated and enantiomeric substrates toward facilitated enzymatic syntheses ofboth d- and t-sugars [80,81]. High stereoselectivity of the wild-type enzyme has been utilized in the preparation of compounds (23) / (24) and in a two-step enzymatic synthesis of (22), the N-terminal amino acid portion of nikkomycin antibiotics (Figure 10.12) [82]. 

This model clearly shows that the catalytic machinery involves a dyad of histidine and aspartate together with the oxyanion hole. Hence, it does not involve serine, which is the key amino acid in the hydrolytic activity of lipases, and, together with aspartate and histidine, constitutes the active site catalytic triad. This has been confirmed by constructing a mutant in which serine was replaced with alanine (Serl05Ala), and finding that it catalyzes the Michael additions even more efficiently than the wild-type enzyme (an example of induced catalytic promiscuity ) [105]. 

The most extensively investigated HNL structures are those from H. brasiliensis (HbHNL)" " and M. esculenta (MeHNL)," which are highly homologous (76% identity). For MeHNL, the crystal structure of the wild-type enzyme complexed with acetone has been reported in 2001 (Fig. 1)." ... 

The CD spectrum of the C188S mutant is essentially the same as that of the wild-type enzyme, which reflects that the tertiary structure of this mutant changed little compared to that of the wild-type enzyme. Calculation of the content of secondary structure of the mutant enzyme based on J-600S Secondary Structure Estimation system (JASCO) also showed that there is no significant change in the secondary structure of the mutant. The fact that the k value of this mutant is extremely small despite little change in conformation clearly indicates that Cysl88 is located in the active site. 

Mattel et al., this book). The three variant enzymes were still able to interact and bind to PGIP with association constants comparable to that of the wild type enzyme. 

Apart from mode of action and kinetics of wild type enzymes structure function relationships of these industrially important enzymes is of high interest to provide the necessary knowledge for genetic engineering of desired properties. As a first approach the identification of catalytically important residues was addressed in conjunction with the elucidation of the three dimensional structure [15]. 

Figure 4. Relative activities of wild type PGII and His223Ala mutated enzyme as a function of pH. Wild type enzyme, solid circles His223Ala mutated enzyme, open circles.

The mutation of ThrlSl, Glyl82, or Glul83 to alanine, or of Glul83 to glutamine also completely inhibited the ATP or acetylphosphate-dependent Ca transport, without effect on the phosphorylation of the enzyme by ATP in the presence of Ca or by Pi in the absence of Ca [127]. The phosphoenzyme formed from ATP retained its ADP-sensitivity at low concentration and alkaline pH, but its rate of decomposition was much slower than that of the wild-type enzyme in the presence of EGTA. These observations implicate the 181-183 region in the conformational changes related to Ca translocation. 

In the case of L-rhamnulose-1-phosphate aldolase (RhaD), we found that the problem of phosphorylated substrate requirement (dihydroxyactone phosphate (DHAP)) could be overcome by a simple change in buffer. Thus, when using borate buffer, reversible borate ester formation created a viable substrate out of dihydroxyacetone, which is not otherwise accepted by the wild-type enzyme (Figure 6.6) [23]. The process was used in a one-step synthesis of... 

The behaviour of the mutant enzymes where, for example, histidine-152 has been changed to alanine is compared with that of wild type enzymes.60 The 31P NMR chemical shift values and signal width for H152A mutant enzyme have shown the presence of two conformers open and closed forms of the enzyme that interconvert slowly on the NMR time scale. The tightness of the binding of the cofactor to the protein surface and its protonation state have been also discussed for intermediate Schiff bases in different steps of the catalytic cycle (Table 1). 

H.A. Azab, L. Banci, M. Borsari, C. Luchinat, M. Sola, and M.S. Viezzoli, Redox chemistry of superoxide dismutase. Cyclic voltammetry of wild-type enzymes and mutants on functionally relevant residues. Inorg, Chem. 31, 4649-4655 (1992). 

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