I. Misra et Hm. Miziorko, EVIDENCE FOR THE INTERACTION OF AVIAN 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE HISTIDINE-264 WITH ACETOACETYL-COA, Biochemistry, 35(29), 1996, pp. 9610-9616
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.