Substrate and inhibitor specificity of 3-hydroxy-3-methylglutaryl-CoA reductase determined with substrate-analogue...

1) Analogues of 3-hydroxy-3-methylglutaryl-CoA were prepared in which the substituents at C-3 of the acyl residue were altered. The same analogues were additionally modified by replacement of the thioester oxygen by hydrogen to yield reduction-resistant CoA-thioethers. The interaction of both types of CoA derivatives with a 58-kDa catalytic fragment of human 3-hydroxy-3-methylglutaryl-CoA reductase was studied. 2) This enzyme reduces glutaryl-CoA at a very low rate whereas 3-hydroxyglutaryl-CoA is well reduced, the maximal rate of reduction being 7% that of the physiological substrate. Only half of total 3-hydroxyglutaryl-CoA was attacked, thus reflecting the stereo-specificity of the enzyme for (3S)-3-hydroxy-3-methylglutaryl-CoA. The results invalidate the hitherto assumed absolute substrate specificity of the enzyme. 3) The affinity of both 3-hydroxyglutaryl-CoA and its thioether variant S-(4-carboxy-3-hydroxybutyl)CoA to the reductase, Ki = 0.3 microM and Ki = 0.4 microM, respectively, is higher than that of the physiological substrate, Km = 1.5 microM (data related to (S)-diastereomer). The results show for the first time that the methyl-group effect observed with the inhibitor lovastatin is an intrinsic property of the enzyme. 4) All of the prepared CoA derivatives are purely competitive inhibitors of the reductase, the affinities varying within a range of two powers of ten (Ki = 0.3-32 microM). On variation of the substituents at C-3 of the acyl residue of the physiological substrate the affinity of both CoA-thioesters and CoA-thioethers increases in the sequence CH2, C(CH3)2, CH(CH3), C(OH)CH3, CH(OH).