EVIDENCE FOR THE INTERACTION OF AVIAN 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE HISTIDINE-264 WITH ACETOACETYL-COA

Previous work on HMG-CoA synthase has implied the presence of a reacti ve active site histidine, prompting our examination of the possible fu nction of invariant histidine residues by site-directed mutagenesis. M utations encoding H197N, H264N/A, and H436N HMG-CoA synthases were con structed, and the mutant enzymes were overexpressed in Escherichia col i BL21(DE3). Kinetic characterization of the isolated synthase variant s indicates that, while H197N and H436N enzymes behave similarly to wi ld-type synthase, H264N and H264A synthases exhibit significant differ ences. Although the K-m for acetyl-CoA is not substantially altered, H 264N/A synthases catalyze production of HMG-CoA at a diminished (simil ar to 25-fold slower) rate. In contrast, H264N/A synthases can efficie ntly catalyze the acetyl-CoA hydrolysis partial reaction exhibiting K- m for acetyl-CoA that, again, approximates the value obtained with the wild-type enzyme. These mutants also retain the ability to form signi ficant levels of the acetyl-S-enzyme reaction intermediate. The functi onal catalysis of partial reactions argues that the H264 mutant protei ns retain substantial structural integrity. In this context, it appear s significant that the H264N/A synthases exhibit a similar to 100-fold increase in the K-m for acetoacetyl-CoA. In order to test whether the two orders of magnitude effect may be largely attributed to a decreas ed affinity of acetoacetyl-CoA for these enzymes and, more specificall y, whether H264 interacts with the carbonyl oxygen of acetoacetyl-CoA' s thioester, turnover of S-(3-oxobutyl)-CoA, a thioether analog of ace toacetyl-CoA, was investigated, This alternative substrate, in which a methylene group replaces the thioester carbonyl, is utilized by wild- type synthase with an apparent V-max that is similar to 100-fold lower and an apparent K-m that is 25-fold higher than the values obtained u sing the physiological substrate, acetoacetyl-CoA. H264A synthase also catalyzes the turnover of S-(3-oxobutyl)-CoA; the diminution in rate supported by the alternative substrate is comparable in magnitude to t he effect observed for wild-type-enzyme. In contrast, H264A exhibits c omparable apparent K-m values for S-(3-oxobutyl)-CoA and acetoacetyl-C oA, Thus, unlike wild-type synthase, there is no penalty in terms of e fficiency of H264A saturation when the alternative thioether substrate replaces the physiological substrate. These data suggest that the imi dazole of H264 in avian enzyme may play a role in anchoring the second substrate, acetoacetyl-CoA, by interacting with the carbonyl oxygen o f the thioester functionality.