Reconstitution of lactic dehydrogenase. Noncovalent aggregation vs. reactivation. 1. Physical properties and kinetics of aggregation

Abstract

The reconstitution of lactic dehydrogenase after dissociation in 6 M guanidine hydrochloride or at acidic pH leads back to active tetramers indistinguishable from the native enzyme. In addition, aggregates of "irreversibly denatured" enzyme are formed to a certain extent. It has been shown that the ratio of both fractions depends on such factors as the extent of denaturation and the concentration of the enzyme in the process of reconstitution. The electron microscopical analysis of the aggregates shows a broad distribution of high molecular weight particles ( M , >lo6). As suggested by circular dichroism measurements in the far-UV and by degradation in strong denaturants, these particles are composed of individual monomeric chains with partially restored secondary structure. The aggregates are stabilized by noncovalent interactions. For determination of the factors responsible for the competition %e great majority of regulatory enzymes represents oligomers composed of subunits which are attached to each other by noncovalent interactions. As a consequence, reconstitution of oligomeric enzymes after dissociation with denaturants, such as guanidine hydrochloride or acid, involves folding and association. In the case of monomeric enzymes, the folding transitions occurring during the formation of the three-dimensional structure have been analyzed in great detail by denaturation-renaturation studies in vitro (Anfinsen & Scheraga, 1975). On the other hand, the reactions involved in oligomer formation are far less characterized (Jaenicke, 1979).The acquisition of the quaternary structure of a given enzyme comprises highly specific association reactions which must be subtly interconnected with folding transitions. Like the unfolding of monomeric enzymes, the dissociation and unfolding of oligomers have been shown to be reversible in many cases. Therefore, the parameters determining the in vitro refolding and reassociation can be analyzed in a quantitative way by transferring denatured and dissociated enzymes to quasi-physiological conditions. Under optimum conditions of denaturation-renaturation, yields close to 100% can be achieved, corresponding to complete reassociation, refolding, and reactivation to the native state of the enzyme. It has been previously shown for a number of enzymes that the product of reconstitution is indistinguishable from the initial native enzyme as far as the hydrodynamic, spectroscopic, and enzymatic properties are concerned (Jaenicke et al., 1975. In cases where only partial reactivation is observed, the latter statement holds only if "irreversibly denatured" material is separated from the native enzyme . No of reactivation and aggregate formation, the kinetics of aggregation were analyzed by stopped-flow laser light scattering experiments and compared with the kinetics of reactivation [cf. Rudolph, R., & Jaenicke, R. (1976) Eur. J. Biochem. 63, 409-4171. Aggregation is determined by a process with a reaction order greater than 2, competing with a fast first-order folding reaction in the path...

Tetrameric and Octameric Lactate Dehydrogenase from the Hyperthermophilic Bacterium Thermotoga maritima

“…As mentioned, denaturation transitions at high temperature or in the presence of chaotropic agents, are perturbed by heat coagulation or kinetic partitioning between unfolding/refolding and aggregation (Zettlmeissl et al, 1979). In Fig.…”

Protein refolding by pH-triggered chaperone binding and release

“…Slow release may provide a convenient way to keep the concentration of aggregation-sensitive folding intermediates low at any given time. Because aggregation is strongly dependent on the concentration of aggregation-sensitive folding intermediates (23), any process that decreases the concentration of these intermediates should greatly decrease the extent of aggregation. In contrast, in the absence of HdeA, pH neutralization may quickly generate a large population of aggregation-sensitive intermediates, which favors their partitioning into the aggregation pathway instead of the refolding pathway.…”

“…Upon pH neutralization, the substrates are released from HdeA at a rate which is very slow compared to the rate of folding to productive, aggregation-resistant intermediates and/or the native state. Because the rate of aggregation (k agg ) is a second or higher order process (23), the decrease in S U concentration dictated by slow release from HdeA following neutralization effectively suppresses aggregation and favors on-pathway substrate-protein refolding.…”

Association-induced folding of globular proteins

“…The storage and delivery of protein drugs are also often complicated by the association process. Protein refolding is often also accompanied by aggregation, especially at higher protein concentrations, which has been attributed to the association of partially folded intermediates (6)(7)(8)(9)(10)(11).…”