The Influence of Conserved Aromatic Residues in 3-Hydroxy-3-methylglutaryl-CoA Synthase

Ila Misra,Chang-Zeng Wang,Henry M Miziorko

doi:10.1074/jbc.m300244200

Ila Misra, Chang-Zeng Wang + Show 1 more

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https://doi.org/10.1074/jbc.m300244200

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Abstract

In order to evaluate the potential contribution of conserved aromatic residues to the hydrophobic active site of 3-hydroxy-3-methylglutaryl-CoA synthase, site-directed mutagenesis was employed to produce Y130L, Y163L, F204L, Y225L, Y346L, and Y376L proteins. Each mutant protein was expressed at levels comparable with wild-type enzyme and was isolated in highly purified form. Initial kinetic characterization indicated that F204L exhibits a substantial (>300-fold) decrease in catalytic rate (kcat). Upon modification with the mechanism-based inhibitor, 3-chloropropionyl-CoA, or in formation of a stable binary complex with acetoacetyl-CoA, F204L exhibits binding stoichiometries comparable with wild-type enzyme, suggesting substantial retention of active site integrity. Y130L and Y376L exhibit inflated values (80- and 40-fold, respectively) for the Km for acetyl-CoA in the acetyl-CoA hydrolysis partial reaction; these mutants also exhibit an order of magnitude decrease in kcat. Formation of the acetyl-S-enzyme reaction intermediate by Y130L, F204L, and Y376L proceeds slowly in comparison with wild-type enzyme. However, solvent exchange into the thioester carbonyl oxygen of these acetyl-S-enzyme intermediates is not slow in comparison with previous observations for D159A and D203A mutants, which also exhibit slow acetyl-S-enzyme formation. The magnitude of the differential isotope shift upon exchange of H218O into [13C]acetyl-S-enzyme suggests a polarization of the thioester carbonyl and a reduction in bond order. Such an effect may substantially contribute to the upfield 13C NMR shift observed for [13C]acetyl-S-enzyme. The influence on acetyl-S-enzyme formation, as well as observed kcat (F204L) and Km (Y130L; Y376L) effects, implicate these invariant residues as part of the catalytic site. Substitution of phenylalanine (Y130F, Y376F) instead of leucine at residues 130 and 376 diminishes the effects on catalytic rate and substrate affinity observed for Y130L and Y376L, underscoring the influence of aromatic side chains near the active site.

Highlights

The condensation of substrates acetyl-CoA and acetoacetylCoA (AcAc-CoA) to form HMG-CoA may be viewed as a threestep process (3) involving formation of an acetyl-S-enzyme intermediate, condensation to form a transient enzyme-SHMG-CoA intermediate, and hydrolysis to release product HMG-CoA (Scheme 1)
In order to evaluate the potential contribution of conserved aromatic residues to the hydrophobic active site of 3-hydroxy-3-methylglutaryl-CoA synthase, site-directed mutagenesis was employed to produce Y130L, Y163L, F204L, Y225L, Y346L, and Y376L proteins
In order to eliminate side chain aromaticity without changing the hydrophobicity, plasmids encoding leucine substitutions for each of these aromatic residues were constructed by mutagenesis of the pET-3d-derived plasmid that encodes wild-type avian HMG-CoA synthase

Summary

Introduction

The condensation of substrates acetyl-CoA and acetoacetylCoA (AcAc-CoA) to form HMG-CoA may be viewed as a threestep process (3) involving formation of an acetyl-S-enzyme intermediate, condensation to form a transient enzyme-SHMG-CoA intermediate, and hydrolysis to release product HMG-CoA (Scheme 1). Evaluation of invariant residues implicated as active site residues a histidine (7) and several acidic residues that influence either formation of the acetyl-S-enzyme intermediate (8) or condensation of this intermediate with the second substrate (9). Model studies and molecular orbital calculations (11) suggested that the effect was potentially attributable to stacking of metabolite with the heterocyclic flavin cofactor of acyl-CoA dehydrogenase. These observations suggested the potential influence of hydrophobic, aromatic residues at the active site of HMG-CoA synthase and prompted evaluation of the importance of aromatic residues that are conserved in this protein. MO). [1,2-13C]Acetic anhydride, used for the production of [1,2-13C]acetyl-CoA (13), was a product of Isotech

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