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Isocitrate Dehydrogenase from Azotobacter vinelandii. Order of Substrate Addition and Product Releaset Jeffrey S. Wicken,$ Albert E. Chung, and James S. Franzen*
: The TPN-specific isocitrate dehydrogenase from Azotobacter cinelandii has been subjected to steady state kinetics analysis. The kinetics of the uninhibited reaction and product inhibition patterns indicate that the forward direction substrates add in a rapid equilibrium random manner and that the products are released predominantly in the order of C 0 2 , 2-ketoglutarate, and TPNH. The system is complicated, under appropriate conditions, by the formation of two deadend complexes, one of the enzyme with isocitrate and TPNH, and the other with T P N + and 2-ketoglutarate. Directly deterABSTRACT
T
he TPN1-specific isocitrate dehydrogenase from Azorohacter cinelandii has been shown to be a monomeric enzyme having a molar mass of 80,000 daltons and containing three sulfhydryl groups of varying reactivity (Chung and Franzen, 1969). It catalyzes the transfer of hydrogen from the substrate, threo-D,-isocitrate, with A type TPN specificity and the decarboxylation of the substrate with retention of configuration (Chung and Franzen, 1970). The most reactive sulfhydryl group is not directly involved in the catalytic process, since it can be quantitatively converted to a thiocyanate group with the resulting derivatized enzyme possessing about 40% of the native specific activity (Chung et al., 1971). The order of substrate addition in the reverse direction of TPNH first, followed successively by 2-ketoglutarate and either HC03- or C 0 2 , has been implicated (Chung and Franzen, 1970). Thompson and Cleland have carried out product inhibition studies on the TPN-specific isocitrate dehydrogenase from pig heart and have concluded that a mechanism involving random substrate addition and ordered product release in the sequence of COY,2-ketoglutarate, and TPNH was likely (Thompson and Cleland, 1965). Colman and Chu have reported that isocitrate and 2-ketoglutarate bind to this pig heart enzyme with respective dissociation constants of 2.25 and 4.62 p~ (Colman and Chu, 1970). Using a fluorescence technique, Langan determined the dissociation constants for the binary complex of TPNH and TPN- with TPN-specific isocitrate dehydrogenase from pig heart homogenates to be on the order of 0.01 and 2.0 p ~ respectively , (Langan, 1960). Since the dissociation constant for the complex of enzyme and 2-ketoglutarate is
t From the Department of Biochemistry of the Faculty of Arts and Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15213. Keceirerl J i d j 17, 1972. Supported in part by grants from the National Institutcs of Health (AM12104 and AM14290). 1 Supported by a National Science Foundation predoctoral stipend. This material \+as submitted in uartial fulfillment of the Ph.D. degree i n biochemistry at the University of Pittsburgh (1971). Present address: Departlnent of Chemistry, Arizona State Cniversit), Tempe, Ariz. I Abbreviations used are: T P N , triphosphopyridinc nucleotide: T P N H , reduced triphosphopyridinc nucleotide; I pK:,'. The slope will be less than unity, but not less than zero, in the intermediate p H range if pK, < pK,'. On the other hand, if noncovalent association occurs the slope of the pKc0, L'S. p H plot has zero slope at low and high pH and positive or negative slope in between depending on whether pK, is greater than pK,," or cice cersa. As is observed in Figure 10, the response of pKCo2 to pH is such as to suggest that CO. adds to the enzyme without the formation of a carbamate derivative and that the process operates essentially like the Bohr effect, that is, a proton is loosened when the CO, adduct forms. As was indicated above, no firm statement can be made about the magnitude of the pK, values though, provisionally, Figure 10 implies that pKz is in the vicinity of 7.5 and pK,' may be about 6.5. In summary, steady state velocity studies in the absence and presence of product inhibitors support the conclusion that A . cinelundii isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate primarily cia the random addition of substrates and the sequential release of products in the order of molecular CO,, 2-ketoglutarate, and TPNH. A minor route of product release involves the inversion of the departure of the last two products from the enzyme. Finally, the existence of the two dead-end complexes, enzyme-isocitrate-TPNH and enzyme-2-ketoglutarate--TPN+, is demonstrated. Referencrs Adkins, B. J., andYang, J. T. (1968), Biochemistry 7,266. Alberty, R . A.,~Smith,R. M., and Bock, R . M. (1951), J . Biol. Chem. 193,425.
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