Acid-base catalysis, site-directed mutagenesis

This enzyme, which is relatively stable under reaction conditions, will retain 70% of its activity after 10 days at pH 5 and 25°C. Although it is not yet commercially available, it has been overexpressed in E. coli, making large quantities easily accessible.68 The detailed mechanism of DERA has been determined based on the atomic structure (ca. 1.0 A) combined with site-directed mutagenesis, kinetic, and NMR studies136 (Scheme 5Alb). A proton relay system composed of Lys and Asp appears to activate a conserved active-site water that functions as the critical mediator for proton transfer in acid-base catalysis. 

It has been established, by means of site-directed mutagenesis experiments, that three amino acid residues are essential for the overall catalysis, namely the glycyl radical (Gly734) and two consecutive cysteines at positions 418 and 419. Two mechanisms were proposed for PFL at the time when the theoretical study was performed. Although based on the same experimental information, these two mechanisms are quite different (Figures 10 and 11). 

There is extensive evidence from site-directed mutagenesis and other studies of enzymes that catalyze proton transfer that acidic and basic amino side chains and, in some cases, metal cations, are required for the observation of efficient catalysis. However, catalysis of the deprotonation of a-carbonyl by small molecule analogs of these side chains, and by metal cations is generally weak. Relatively little attention has been directed towards understanding the mechanism for the enhancement of Bronsted acid/base and electrophilic catalysis for enzyme-catalyzed reactions... 

In several cases, site-directed mutagenesis has helped to identify residues directly involved in acid-base catalysis. Deletion of residues which participate in acid-base catalysis leads to a marked reduction in catalytic activity at neutral pH and an alteration in the pH dependence for catalysis similar to results described... 

Catalysis by (6—4) photolyase must accomplish two chemical tasks cleavage of the C6—C4 sig a bond, and transfer of the OH (or —NH2) group from the C5 of the 5 base to the G4 of the 3 base. Because formation of the (6—4) photoproduct is presumed to proceed through a four-mem-bered oxetane or azetidine intermediate, it has been proposed that (6—4) photolyase first converts the open form of the (6—4) photoproduct to the four-membered ring by a thermal reaction, and then the four-mem-bered ring is cleaved by retro [2+2] reaction photochemically (Kim et al, 1994 Zhao et aL, 1997). A site-directed mutagenesis study has identified two histidine residues in the active site that may participate in conversion of the (6-4) photoproduct to the oxetane intermediate by general acid-base catalysis (Hitomi et al, 2001). A current model for catalysis by (6-4) photolyase is as follows (Fig. 8) The enzyme binds DNA and flips out the... 

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