TAILIEUCHUNG - Báo cáo Y học: Recombinant human glucose-6-phosphate dehydrogenase Evidence for a rapid-equilibrium random-order mechanism

Cloning and over-expression of human glucose 6-phosphate dehydrogenase (Glc6P dehydrogenase) has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme, studied by fluorimetric initial-rate measurements, gave converging linear Lineweaver–Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP+ and Glc6P separately to form binary complexes, suggesting a random-order mechanism | Eur. J. Biochem. 269 3417-3424 2002 FEBS 2002 doi Recombinant human glucose-6-phosphate dehydrogenase Evidence for a rapid-equilibrium random-order mechanism Xiao-Tao Wang1 Shannon W. N. Au1 2 Veronica M. S. Lam1 w and Paul C. Engel 1 Department of Biochemistry The University of Hong Kong Hong Kong SAR 2Section of Structural Biology Institute of Cancer Research Chester Beatty Laboratory London UK 3Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research University College Dublin Ireland Cloning and over-expression of human glucose 6-phosphate dehydrogenase Glc6P dehydrogenase has for the first time allowed a detailed kinetic study of a preparation that is genetically homogeneous and in which all the protein molecules are of identical age. The steady-state kinetics of the recombinant enzyme studied by fluorimetric initial-rate measurements gave converging linear Lineweaver-Burk plots as expected for a ternary-complex mechanism. Patterns of product and dead-end inhibition indicated that the enzyme can bind NADP and Glc6P separately to form binary complexes suggesting a random-order mechanism. The Ki value for the binding of NADP measured by titration of protein fluorescence is M close to the value of M calculated from the kinetic data on the assumption of a rapid-equilibrium random-order mechanism. Strong evidence for this mechanism and against either of the compulsory-order possibilities is provided by repeating the kinetic analysis with each of the natural substrates replaced in turn by structural analogues. A full kinetic analysis was carried out with deaminoNADP and with deoxyglucose 6-phosphate as the alternative substrates. In each case the calculated dissociation constant upon switching a substrate in a random-order mechanism . that for NADP upon changing the sugar phosphate was indeed constant within experimental error as expected. The calculated rate constants for binding of the .

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