Detection of multiple active site domain motions in transient-state component time courses of the Clostridium symbiosum L-glutamate dehydrogenase-catalyzed oxidative deamination reaction.
We present a multiwavelength, transient-state kinetic study of the oxidative deamination reaction catalyzed by Clostridium symbiosum glutamate dehydrogenase (csGDH) producing the real-time reaction courses of spectroscopically resolved kinetically competent intermediate complexes. The results show striking differences from a corresponding transient-state study of the same reaction by the structurally homologous enzyme from beef liver (blGDH). In addition to the highly blue-shifted alpha-iminoglutarate and highly red-shifted carbinolamine complexes observed in both reactions, the csGDH reaction appeared to show the release of free NADH at a very early and mechanistically unlikely point in the reaction. Using lactic acid dehydrogenase as a "reporter" for free NADH, we show that the early portion of this signal reflects previously unobserved spectrally unshifted enzyme-bound NADH complexes. We provide experimental evidence to show that such spectrally anomalous complexes must represent forms of the known alpha-imino and alpha-carbinolamine complexes in which the active site cleft is open. This evidence includes isothermal calorimetric measurements and pH-jump experiments that show the existence of differing two-state transitions in blGDH and csGDH and locate active site domain motions at differing points in the transient-state time courses of the two enzyme reactions. We prove the kinetic competence of a new and more highly detailed mechanism for the csGDH reaction that involves the alternation of open and closed enzyme complexes as integral steps. These findings, supported by the available X-ray crystal structure data, suggest the existence of a programmed time course of protein domain motions coordinated with the classically considered chemical time course. This new viewpoint may be presumed to be applicable to enzyme reactions other than those of the alpha-amino acid dehydrogenases.
Tally
Jon F
JF
Laboratory of Molecular Biochemistry, Veteran Affairs Medical Center, and Department of Biochemistry, University of Kansas Medical Center, Kansas City, Missouri 64128, USA.
Maniscalco
Steven J
SJ
Saha
Swapan K
SK
Fisher
Harvey F
HF
D000586:Amination; D000818:Animals; D001665:Binding Sites; D002151:Calorimetry; D002384:Catalysis; D002417:Cattle; D003013:Clostridium / Q000201:enzymology*; D003903:Deuterium; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D008956:Models, Chemical; D009243:NAD / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D013816:Thermodynamics
D009243:NAD; D003903:Deuterium; D005969:Glutamate Dehydrogenase; C061421:glutamate dehydrogenase (NAD(P)+)