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. The Kd value for the binding of NADP+ measured by titration of protein fluorescence is 8.0 microm, close to the value of 6.8 microm 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 (e.g. that for NADP+ upon changing the sugar phosphate) was indeed constant within experimental error as expected. The calculated rate constants for binding of the leading substrate in a compulsory-order mechanism, however, did not remain constant when the putative second substrate was changed. Previous workers, using enzyme from pooled blood, have variously proposed either compulsory-order or random-order mechanisms. Our study appears to provide unambiguous evidence for the latter pattern of substrate binding.
Wang
Xiao-Tao
XT
Department of Biochemistry, The University of Hong Kong, Hong Kong SAR.
Au
Shannon W N
SW
Lam
Veronica M S
VM
Engel
Paul C
PC
D005944:Glucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism
D011993:Recombinant Fusion Proteins; C001293:glucosamine 6-phosphate; D009249:NADP; D019298:Glucose-6-Phosphate; D005954:Glucosephosphate Dehydrogenase; D005944:Glucosamine