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Avian 3‐hydroxy‐3‐methylglutaryl‐CoA lyase: Sensitivity of enzyme activity to thiol/disulfide exchange and identification of proximal reactive cysteines
Author(s) -
Hruz Paul W.,
Miziorko Henry M.
Publication year - 1992
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560010908
Subject(s) - chemistry , cysteine , enzyme , phosphofructokinase 2 , thiol , reagent , bifunctional , enzyme assay , substrate (aquarium) , lyase , redox , biochemistry , stereochemistry , catalysis , organic chemistry , biology , ecology
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 k inact of 0.178 min −1 . The oxidized enzyme is inactivated at a sixfold slower rate ( k inact = 0.028 min −1 ). 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 k inact 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‐dibromo‐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 [1‐ 14 C]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 thiol/disulfide exchange and identify a cysteinyl residue with the reactivity and juxtaposition required for participation in disulfide formation.