Modeling of a Mutation Responsible for Human 3-Hydroxy-3-methylglutaryl-CoA Lyase Deficiency Implicates Histidine 233 as an Active Site Residue

Jacqueline R Roberts,Grant A Mitchell,Henry M Miziorko

doi:10.1074/jbc.271.40.24604

Abstract

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is inactivated by diethyl pyrocarbonate (DEPC); activity can be fully restored by incubation with hydroxylamine. Protection against DEPC inactivation is afforded by a substrate analogue, suggesting an active site location for a DEPC target. Included in the inherited defects that map within the HMG-CoA lyase gene is a point mutation that results in an arginine substitution for histidine 233, one of only two invariant histidines. These observations prompted a functional test of the importance of His-233. The mutant lyases H233R, H233A, and H233D were overexpressed in Escherichia coli, isolated, and kinetically characterized. In H233D, DEPC targets one less histidine than was measured using wild-type lyase, supporting the assignment of wild-type lyase His-233 as one of the DEPC targets. Substitution of His-233 results in diminution of activity by approximately 4 orders of magnitude. Km values of the mutant lyases for both substrate HMG-CoA and activator divalent cation (Mg2+ or Mn2+) are comparable to the values measured for wild-type enzyme, indicating that these enzymes retain substantial structural integrity. This conclusion is reinforced by the observation that the affinity label, 2-butynoyl-CoA, stoichiometrically modifies the mutant lyases, indicating that they contain a full complement of active sites. In view of these data suggesting that the structures of these mutant lyases closely approximate that of the wild-type enzyme, their observed 10(4)-fold diminution in catalytic efficiency supports assignment to His-233 of a role in the chemistry of HMG-CoA cleavage.

Highlights

3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) catalyses the cleavage of HMG-CoA1 into acetoacetate and acetylCoA
diethyl pyrocarbonate (DEPC) Inactivation of HMG-CoA Lyase—Upon incubation of HMG-CoA lyase with DEPC (0.25–2.5 mM) in potassium phosphate buffer, pH 6.8, a time-dependent loss of activity was observed (Fig. 1)
When HG-CoA was preincubated with human HMG-CoA lyase, the rate of inactivation by DEPC was greatly reduced (Fig. 4), indicating that the substrate analogue, HG-CoA, was able to afford significant protection. These results strongly suggest that a histidine targeted by DEPC is situated within the active site pocket of HMG-CoA lyase

Summary

Introduction

3-Hydroxy-3-methylglutaryl-CoA lyase (EC 4.1.3.4) catalyses the cleavage of HMG-CoA1 into acetoacetate and acetylCoA. Human HMG-CoA lyase has recently been expressed in Escherichia coli cells and purified to homogeneity [4] Using this expression system, the active site cysteine (Cys-266) of human HMG-CoA lyase was altered by site-directed mutagenesis to determine whether this amino acid was a key component of the catalytic apparatus [5]. In the course of our ongoing studies of mutations in HMG-CoA lyasedeficient patients [7, 10, 11], we discovered a missense mutation in the codon of the highly conserved His-233 residue.2 This information prompted our investigation of the sensitivity of HMG-CoA lyase to histidine modification, which, in turn, suggested that a test of the function of His-233 by directed mutagenesis would be informative. As documented in this report, the results of these studies implicate His-233 in catalysis of HMG-CoA cleavage

Methods

Results

Conclusion