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 reactive active site histidine, prompting our examination of the possible function of invariant histidine residues by site-directed mutagenesis. Mutations encoding H197N, H264N/A, and H436N HMG-CoA synthases were constructed, and the mutant enzymes were overexpressed in Escherichia coli BL21(DE3). Kinetic characterization of the isolated synthase variants indicates that, while H197N and H436N enzymes behave similarly to wild-type synthase, H264N and H264A synthases exhibit significant differences. Although the k(m) for acetyl-CoA is not substantially altered, H264N/A synthases catalyze production of HMG-CoA at a diminished (approximately 25-fold slower) rate. In contrast, H264N/A synthases can efficiently catalyze the acetyl-CoA hydrolysis partial reaction exhibiting a k(m) for acetyl-CoA that, again, approximates the value obtained with the wild-type enzyme. These mutants also retain the ability to form significant levels of the acetyl-S-enzyme reaction intermediate. The functional catalysis of partial reactions argues that the H264 mutant proteins retain substantial structural integrity. In this context, it appears significant that the H264N/A synthases exhibit a approximately 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 decreased affinity of acetoacetyl-CoA for these enzymes and, more specifically, whether H264 interacts with the carbonyl oxygen of acetoacetyl-CoA's thioester, turnover of S-(3-oxobutyl)-CoA, a thioether analog of acetoacetyl-CoA, was investigated. This alternative substrate, in which a methylene group replaces the thioester carbonyl, is utilized by wild-type synthase with an apparent Vmax that is approximately 100-fold lower and an apparent k(m) that is 25-fold higher than the values obtained using 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 the effect observed for wild-type enzyme. In contrast, H264A exhibits comparable apparent k(m) values for S-(3-oxobutyl)-CoA and acetoacetyl-CoA. Thus, unlike wild-type synthase, there is no penalty in terms of efficiency of H264A saturation when the alternative thioether substrate replaces the physiological substrate. These data suggest that the imidazole of H264 in avian enzyme may play a role in anchoring the second substrate, acetoacetyl-CoA, by interacting with the carbonyl oxygen of the thioester functionality.