Avian 3‐hydroxy‐3‐methylglutaryl‐CoA lyase: Sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines

Abstract

Catalysis by purified avian 3-hydroxy-3-methylglutaryl-CoA lyase is critically dependent on the reduction state of the enzyme, with less than 1% of optimal activity being observed with the air-oxidized enzyme. The enzyme is irreversibly inactivated by sulfhydryl-directed reagents with the rate of this inactivation being highly dependent upon the redox state of a critical cysteine. Methylation of reduced avian lyase with 1 mM 4-methylnitrobenzene sulfonate results in rapid inactivation of the enzyme with a kino,., of 0. I78 min" . The oxidized enzyme is inactivated at a sixfold slower rate (kino,, = 0.028 min-I). Inactivation of the enzyme with the reactive substrate analog 2-butynoyl-CoA shows a similar dependence upon the enzyme's redox state, with a sevenfold difference in kin,,, observed with oxidized vs. reduced forms of the enzyme. Chemical cross-linking of the reduced enzyme with stoichiometric amounts of the bifunctional reagents 1,3-dibrom0-2-propanone (DBP) or N, N-ortho-phenylenedimaleimide (PDM) coincides with rapid inactivation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of enzyme treated with bifunctional reagent reveals a band of twice the molecular weight of the lyase monomer, indicating that an intersubunit cross-link has been formed. Differential labeling of native and cross-linked protein with [l-14C]iodoacetate has identified as the primary cross-linking target a cysteine within the sequence VSQAACR, which maps at the carboxy-terminus of the cDNA-deduced sequence of the avian enzyme (Mitchell, G.A., et al., 1991, Am. J. Hum. Genet. 49, 101). In contrast, bacterial HMG-CoA lyase, which contains no corresponding cysteine, is not cross-linked by comparable treatment with bifunctional reagent. These results provide evidence for a potential regulatory mechanism for the eukaryotic enzyme via thioVdisulfide exchange and identify a cysteinyl residue with the reactivity and juxtaposition required for participation in disulfide formation.

Ten novelHMGCLmutations in 24 patients of different origin with 3-hydroxy-3-methyl-glutaric aciduria

“…The HL assay (Wanders et al, 1988) was performed in reductive conditions because the enzyme was more active than in oxidative conditions (Roberts et al, 1994;Hruz and Miziorko, 1992). Although the enzyme in nature is a dimer linked by disulfide bonds, in this redox state HL is a monomer, indicating that there is no protein-protein interaction that might modify the activity values.…”

Crystal Structures of Two Bacterial 3-Hydroxy-3-methylglutaryl-CoA Lyases Suggest a Common Catalytic Mechanism among a Family of TIM Barrel Metalloenzymes Cleaving Carbon-Carbon Bonds

“…An inter-dimer contact of this kind in a disulfide-linked tetramer of hsHL could either constrain the G-loop in a catalytically inefficient conformation or propagate an activity-attenuating allosteric conformational change into the active site of the enzyme. Thus, oxidative crosslinking in the common tetrameric assembly observed in the crystal structures of bsHL and hsHL could play a role in the attenuation of hsHL activity under oxidative stress conditions in human cells (11). (45,46) showing just helices ␣12 and residues Lys-297 in the tetramer interface.…”

“…The residue occurring at the equivalent position in hsHL is Cys-323 (Fig. 2), and this residue is known to form a disulfide bond with the same residue in another subunit under oxidizing conditions, a reaction that strongly attenuates enzyme activity (11). The S-␥ atoms from this residue in subunits from two different dimers come within 5.5 Å of one another in the tetrameric assembly found in the crystal structure of bsHL (Fig.…”

“…The dashed lines connect the C-␥ atoms of this residue in subunits A and B to the equivalent atoms in subunits C and D, respectively, with the corresponding distances indicated in Å. This atom corresponds to the S-␥ atom of residue Cys-323 in hsHL, which occurs at the equivalent position and forms a disulfide bond during oxidative stress (11).…”

“…Oxidative stress down-regulates hsHL activity via formation of a stable intersubunit disulfide linkage. Although the mechanistic basis of the resulting reduction in enzyme activity is not understood, the disulfide bond holding this complex together is believed to be a symmetrical linkage between pairs of Cys-323 residues (11).…”