Metabolite-modulated complex formation between .alpha.-glycerophosphate dehydrogenase and lactate dehydrogenase

Abstract

A modified Hummel-Dreyer equilibrium chromatography technique was used to test the hypothesis that NADH induces the molecular association of lactate dehydrogenase (LDH) and alpha-glycerol-3-phosphate dehydrogenase (alpha-GDH). In the presence of a very limited NADH concentration, a unique elution profile with a new peak running immediately ahead of a trough at the free alpha-GDH elution position is obtained. The appearance of this peak-trough profile is physical evidence that reversible association between LDH and alpha-GDH occurs over a very limited range of free NADH concentrations. The association constant for this complex formation between LDH and alpha-GDH is estimated to be 2.0 microM-1. With the NADH concentration increased to saturation level, no evidence of binding is observed. Such concentration-dependent behavior suggests that the strong competition between LDH and alpha-GDH for the limited amount of NADH tends to promote the enzyme-enzyme contact in order to make the most efficient use of the shared metabolite. The experimental results described in this article make a convincing argument for a metabolite-modulated enzyme-enzyme interaction along this metabolic pathway.

Protein-protein interactions: methods for detection and analysis.

“…The CheW concentration in the sample at which there was no resulting CheW peak or trough represented a sample at true equilibrium. From this, they could calculate a dissociation constant of the complex of 13 M. A similar series of experiments was done by Yong et al (243) to demonstrate an interaction between glycerol-3-phosphate dehydrogenase and lactate dehydrogenase over an extremely limited range of NADH concentrations. Such a complex was observed only when the NADH concentration was high enough for an interaction and low enough to be shared by the two enzymes, and it provided evidence for substrate channeling.…”

New experimental approaches for investigating interactions between Pyrococcus furiosus carbamate kinase and carbamoyltransferases, enzymes involved in the channeling of thermolabile carbamoyl phosphate

“…The influence of CP (and other substrates) on complex formation is worth further investigation. Using Hummel-Dreyer equilibrium chromatography, Yong et al (1993) found a metabolite modulated enzyme-enzyme interaction: the reversible association between lactate dehydrogenase and α-glycerol-3-phosphate dehydrogenase was shown to occur over a limited range of NADH concentrations.…”

Absence of Evidence for Metabolite-Modulated Association between α-Glycerol-3-phosphate Dehydrogenase and l-Lactate Dehydrogenase

“…However, Yong et al (1) reported detecting a strong complex (K a ) 2.0 ( 0.6 µM -1 ) in the presence of a very limited substoichiometric [NADH], a condition not tested in previous studies. The detection of a weak complex (K a ) 0.04 ( 0.02 µM -1 ) in the absence of NADH, but not in the presence of a saturating [NADH], was also reported (1). This NADH-modulated association suggested an unprecedented but reasonable mechanism of enzyme interactions of potentially great importance to enzyme modulation and the controversial phenomena of direct NADH transfer (channeling) between dehydrogenases (for review, ref 4).…”

“…Previous attempts to detect a complex between these two dehydrogenases were unsuccessful (2,3). However, Yong et al (1) reported detecting a strong complex (K a ) 2.0 ( 0.6 µM -1 ) in the presence of a very limited substoichiometric [NADH], a condition not tested in previous studies. The detection of a weak complex (K a ) 0.04 ( 0.02 µM -1 ) in the absence of NADH, but not in the presence of a saturating [NADH], was also reported (1).…”

“…Evidence for the metabolite-modulated formation of an equilibrium complex between R-glycerol-3-phosphate dehydrogenase (RGDH; EC 1.1.1.8) 1 and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was reported (1). Previous attempts to detect a complex between these two dehydrogenases were unsuccessful (2,3).…”