Histidine active formate donor

Since wild-type BFD does not accept ortho-substituted benzaldehydes as donor substrates, the variant BFDH281A with an enlarged active site was prepared by site-directed mutagenesis of histidine-281 to alanine. This variant shows an activity higher by a factor of >140 with respect to the formation of (R)-benzoin. Furthermore, BFDH281A allows the selective formation of mixed substituted (P)-benzoins by employment of appropriate substrate combinations [10]. 

Glycosidic coupling of the acceptor 27 and the donor 23 cleanly provides disaccharide 28, in which the diphenylphosphate activation is clearly preferable to earlier methods. The a-linkage is retained exclusively due to the presence of the C2 acetate, as planned. Activation and coupling of disaccharide 28 with the protected (3-hydroxy-l-histidine derivative 29 gives the expected adduct, with an a/ 3 anomer ratio of at least 13 1. This is probably due to the low reactivity of the glycosyl acceptor, which favors formation of the more stable a-anomer. The adduct is then converted into 30, which is suitably prepared for linking with tetrapeptide S and pyrimidoblamic acid. 

Citrate synthase catalyzes the condensation reaction by bringing the substrates into close proximity, orienting them, and polarizing certain bonds. Two histidine residues and an aspartate residue are important players (Figure 1711). One of the histidine residues (His 274) donates a proton to the carbonyl oxygen of acetyl CoA to promote the removal of a methyl proton by Asp 375. Oxaloacetate is activated by the transfer of a proton from His 320 to its carbonyl carbon atom. The concomitant attack of the enol of acetyl CoA on the carbonyl carbon of oxaloacetate results in the formation of a carbon-carbon bond. The newly formed citryl CoA induces additional structural changes in the enzyme. The active site becomes completely enclosed. His 274 participates again as a proton donor to hydrolyze the thioester. Coenzyme A leaves the enzyme, followed by citrate, and the enzyme returns to the initial open conformation. 

Proposals made concerning this hydrolysis mechanism suggest that the active site of AChE consists of a nucleophilic serine residue and a histidine residue which probably serves as a proton donor or receiver (6). Hydrolysis is thought to occur through the formation of a Michealis complex between the active site of the AChE and the substrate (S), the conversion of the complex in an "acylation" reaction to an acyl enzyme intermediate (AChE-A) and the product choline (or thiocholine), and the subsequent hydrolysis of the acyl enzyme in a "deacylation" reaction to acetic acid and the regenerated enzyme (1,8,9). 

The N-5 position is considerably more basic than the N-10 position, and this basicity is one of several factors that control certain preferences in the course of reactions involving tetrahydrofolate. Thus, for-mylation occurs more readily at N-10 while alkylation occurs more readily at N-5. Benkovic and Bullard (1973) have reviewed evidence for an iminium cation at N-5 as the active donor in formaldehyde oxidation-level transfers. Recently, Barrows et al. (1976) have further studied such a mechanism for folic acid. The interconversion of these forms of folate coenzymes by enzymatic means has been reviewed by Stokstad and Koch (1967), and the reader is directed there for further details. Folate coenzymes are involved in a wide variety of biochemical reactions. These include purine and pyrimidine synthesis, conversion of glycine to serine, and utilization and generation of formate. In addition, the catabolism of histidine, with the formation of formiminoglu-tamic acid (FIGLU), is an important cellular reaction involving folate. 

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