Reassociation of dimeric cytoplasmic malate dehydrogenase is determined by slow and very slow folding reactions

Abstract

Malate dehydrogenase occurs in virtually all eucaryotic cells in mitochondrial and cytoplasmic forms, both of which are composed of two identifical subunits. The reactivation of the mitochondrial isoenzyme has been the subject of previous studies [Jaenicke, R., Rudolph, R., and Heider, I. (1979) Biochemistry 18, 1217-1223]. In the present study, the reconstitution of cytoplasmic malate dehydrogenase from porcine heart after denaturation by guanidine hydrochloride has been determined. The enzyme is denatured by > 1.2 M guanidine hydrochloride; upon reconstitution, .apprx. 60% of the initial native enzyme can be recovered. The kinetics of reconstitution after maximum unfolding by 6 M guanidine hydrochloride were analyzed by fluorescence, fat-ultraviolet circular dichroism, chemical cross-linking with glutaraldehyde, and activity measurements. After fast folding into structure intermediates (< 1 min), formation of native enzyme is governed by two parallel show and very slow first-order folding reactions (k1 = 1.3 .times. 10-3 s-1 and k2 = 7 .times. 10-5 s-1 at 20.degree. C). The rate constant of the association step following the slow folding reaction (determined by k1) must be > 106 M-1 s-1. The energy of activation of the slow folding step is of the order of 9 .+-. 1 kcal/mol; the apparent rate constant of the parallel very slow folding reaction is virtually temperature independent. The intermediates of reassociation must be enzymatically inactive, since reactivation strictly parallels the formation of native dimers. Upon acid dissociation (pH 2.3), .apprx. 35% of the native helicity is preserved, as determined by circular dichroism. Despite the residual structure, reconstitution after short-term acid dissociation (5 min) is governed by the same slow and very slow folding reactions as those observe after unfolding by guanidine hydrochloride. After long-term acid incubation (24 h), both the rate and yield of reactivation decrease, presumably due to a conformational rearrangement within the acid-dissociated monomers.