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 K_m of 13 μM 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 K_m values for Mg²⁺ and Mn²⁺ are dependent upon the substrate used. In contrast, the K_m 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.