Rate-determining folding and association reactions on reconstitution pathway of porcine skeletal muscle lactic dehydrogenase after denaturation by guanidine hydrochloride

Abstract

Reactivation of tetrameric porcine skeletal muscle lactic dehydrogenase after dissociation and extensive unfolding of the monomers by 6 M guanidine hydrochloride (Gdn . HCl) is characterized by sigmoidal kinetics, indicating a complex mechanism involving rate-limiting folding and association steps. For analysis of the association reactions, chemical cross-linking with glutaraldehyde may be used [Hermann, R., Jaenicke, R., & Rudolph, R. (1981) Biochemistry 20, 2195-2201]. The data clearly show that the formation of a dimeric intermediate is determined by a first-order folding reaction of the monomers with k1 = (8.0 +/- 0.1) x 10(-4) s-1. The rate constant of the association of dimers to tetramers which represents the second rate-limiting step on the pathway of reconstitution after guanidine denaturation, was then determined by reactivation and cross-linking experiments after dissociation in 0.1 M H3PO4 containing 1 M Na2SO4. The rate constant for the dimer association (which is the only rate-limiting step after acid dissociation) was k2 = (3.0 +/- 0.5) x 10(4) M-1 s-1. On the basis of the given two rate constants, the complete reassociation pattern of porcine lactic dehydrogenase after dissociation and denaturation in 6 M Gdn . HCl can be described by the kinetic model (formula: see text).

The folding of dimeric cytoplasmic malate dehydrogenase

“…The equilibrium unfolding transitions are fully reversible except for the reactivation transition, which recovers 60% of native state activity. However, this is not an unusual case and has been reported previously in other oligomeric proteins [21,22,42,43]. This is probably because reactivation needs finer tuning of the folding in the active site of the protein than the other transitions.…”

“…Reconstitution was initiated by 200-fold dilution of the denaturation mixture in 100 mM sodium phosphate pH 7.6 at 20°C, so that the residual denaturant concentration was 30 mM (above which no successful crosslinking could occur). Chemical cross-linking with glutaraldehyde was carried out using a method modified from Zettlmissl et al [28]. The cross-linking products were run in SDS/PAGE for separation.…”

“…Chemical cross‐linking with glutaraldehyde was carried out using a method modified from Zettlmissl et al . [28]. The cross‐linking products were run in SDS/PAGE for separation.…”

Extremely thermostable L(+)‐lactate dehydrogenase from thermotoga maritima: Cloning, characterization, and crystallization of the recombinant enzyme in its tetrameric and octameric state

“…The quaternary structure of the enzyme was determined by analytical ultracentrifugation and chemical crosslinking with glutaraldehyde (Hermann et al, 1981;Zettlmeissl et al, 1982). The procedure was optimized regarding glutaraldehyde concentration (1% w/v), protein concentration (1 pg/mL), and the subsequent SDS-PAGE (5-25% acrylamide gels).…”

2 Protein Folding and its Implications for the Production of Recombinant Proteins

“…The kinetic mechanism shows a very rapid monomer-dimer equilibrium, which is close to the rate limit imposed by diffusion (Hermann, Jaenicke and Rudolph, 1981). Dimer formation is determined by folding of the monomers, which is a first-order process (Zettlmeissl, Rudolph and Jaenicke, 1982):…”