3-Hydroxy-3-methylglutaryldithio-CoA: utility of an alternative substrate in elucidation of a role for HMG-CoA lyase's cation activator.
(S)-(3-Hydroxy-3-methyl-1-thionoglutaryl)-Coenzyme A (HMG[= S]CoA), a dithioester analog of (S)-(3-hydroxy-3-methylglutaryl)-CoA (HMG-CoA), acts as an efficient alternative substrate for avian HMG-CoA lyase. Detection of product formation by HPLC, UV absorbance and coupled enzyme assays indicates that HMG[= S]CoA cleavage yields acetyl[= S]CoA and acetoacetate. HMG[= S]CoA binds to the lyase with a Km of 13 microM and undergoes the cleavage reaction at a maximal rate which is 20% of that observed with HMG-CoA. The enzyme-catalyzed cleavage of both HMG-CoA and HMG[= S]CoA is stimulated by the divalent cations Mg2+ and Mn2+. Mg2+ produces a 2-fold higher stimulation of HMG-CoA cleavage than that observed with Mn2+. In contrast, stimulation of HMG[= S]CoA cleavage is nearly seven times higher with Mn2+ than with Mg2+. Not only is the stimulation of enzymatic activity dependent on the cation, but also the Km values for Mg2+ and Mn2+ are dependent upon the substrate used. In contrast, the Km values for HMG-CoA and HMG[= S]CoA are not markedly dependent on the identity of the divalent cation. These results are compatible with the initial formation of a binary enzyme-substrate complex prior to binding of the divalent cation to produce a catalytically active enzyme-substrate-metal ternary complex.
Hruz
P W
PW
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Anderson
V E
VE
Miziorko
H M
HM
D000214:Acyl Coenzyme A / Q000138:chemical synthesis / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001717:Birds; D002413:Cations, Divalent; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006904:Hydroxymethylglutaryl-CoA Synthase / Q000737:chemistry*; D008099:Liver / Q000201:enzymology; D008274:Magnesium; D008345:Manganese; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D002413:Cations, Divalent; C070602:3-hydroxy-3-methylglutaryldithio-coenzyme A; D008345:Manganese; D006904:Hydroxymethylglutaryl-CoA Synthase; D008274:Magnesium