title: Reexamination of the kinetics of the transfer of NADH between its complexes with glycerol-3-phosphate dehydrogenase and with lactate dehydrogenase.
abstract: Srivastava and Bernhard [Srivastava, D. K. & Bernhard, S. A. (1986) Science 234, 1081-1086] have proposed that glycolytic enzymes form multienzyme complexes for the direct transfer of metabolites from the producing enzyme to the utilizing one. We have reinvestigated the evidence for direct transfer of NADH between its complexes with alpha-glycerol-3-phosphate dehydrogenase (GPDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27). The results reveal the following. (i) Proper treatment of the kinetics of and equilibrium data for the transfer of NADH between GPDH and LDH indicates that NADH transfer proceeds by a free-diffusion mechanism and not by direct transfer through a ternary complex. (ii) The koff for NADH from its GPDH complex is 60 sec-1 rather than 9.4 sec-1 in Tris.HCl buffer (pH 7.4) at 25 degrees C. With this value one can explain kcat = 50 sec-1 for LDH-catalyzed hydrogenation of pyruvate with GPDH-bound NADH as coenzyme. (iii) Steady-state kinetics show that LDH inhibits the GPDH-catalyzed reaction simply by reducing the concentration of free NADH. Similarly, aldolase inhibits the GPDH-catalyzed reduction of dihydroxyacetone phosphate to glycerol-3-phosphate by binding to the substrate. The proposed direct transfer of NADH between GPDH and LDH is therefore mainly based on a misinterpretation of the experimental data.
authors.affiliation: Laboratory of Biochemistry, National Institutes of Health, Bethesda, MD 20892.
authors.forename: P B
authors.initials: PB
authors.lastname: Chock
authors.forename: H
authors.initials: H
authors.lastname: Gutfreund
mesh_terms: D000818:Animals; D005993:Glycerolphosphate Dehydrogenase / Q000378:metabolism*; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000378:metabolism*; D009132:Muscles / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD / Q000378:metabolism*; D010084:Oxidation-Reduction; D013050:Spectrometry, Fluorescence
chemical_list: D009243:NAD; D005993:Glycerolphosphate Dehydrogenase; D007770:L-Lactate Dehydrogenase