Mechanism and Specificity of Reconstitution of Dimeric Lactate Dehydrogenase fromLimulus polyphemus

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Abstract
D-Lactate dehydrogenase (EC 1.1.1.28) from L. polyphemus is a homodimer which is composed of identical subunits of MW 35,000. The enzyme may be reversibly denatured and dissociated at acid pH or in 6 M guanidine .cntdot. HCl. The sigmoidal time course of reactivaton obeys a consecutive uni-bimolecular mechanism with k1 = 6 .times. 10-4 s-1 and k2 = 1.3 .times. 104 M-1 s-1 (20.degree. C) as 1st- and 2nd-order rate constants. Cross-linking experiments with glutaraldehyde prove that reactivation and dimer formation run parallel. Joint synchronous reconstitution of the enzyme with dimeric porcine mitochondrial malate dehydrogenase (after denaturation in 6 M guanidine .cntdot. HCl) does not yield active hybrids. The unchanged kinetics of reactivation in the absence and presence of the prospective partner of hybridization prove that inactive hybrid intermediates may also be excluded. The absence of hybrids upon synchronous reconstitution of the 2 closely related dimeric NAD-dependent dehydrogenases clearly suggests that the assembly of nascent oligomeric proteins must be highly specific.