A method for the purification of yeast K+-activated aldehyde dehydrogenase is presented which can be completed in substantially less time than other published procedures. The enzyme has a different N-terminal amino acid from preparations previously reported, and other small differences in amino acid content. These differences may be the result of differential proteolytic digestion rather than a different protein in vivo. A purification step involves the biospecific adsorption on affinity columns containing immobilized nucleotides in the absence of the substrate aldehyde. Direct binding studies with the coenzyme in the absence of aldehyde reveal 4 NAD sites per tetrameric molecule, each with a dissociation constant of 120 micron. These results conflict with properties of preparations previously reported and may conflict with kinetic models that have aldehyde as the leading substrate. Binding to Blue Dextran affinity columns suggests the presence of a dinucleotide fold in common with other dehydrogenases and kinases.

Following the criticism by Chock and Gutfreund [Chock, P. B. & Gutfreund, H. (1988)Proc. Nall. Acad. Sci. USA 85,[8870][8871][8872][8873][8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (aglycerol phosphate dehydrogenase, a-GDH; EC 1.1.1.8) and L-lactate dehydrogenase (LDH; EC 1.1.1.27) was based on a misinterpretation of the kinetic data, we have reinvestigated the transfer mechanism between this enzyme pair. By using the "enzyme buffering" steady-state kinetic technique [Srivastava, D. K. & Bernhard, S. A. (1984) Biochemistry 23, 4538-45451, we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle a-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the a-GDH-NADH complex can serve as substrate for the a-GDH-catalyzed reaction and the LDH-catalyzed reaction, respectively. This is consistent with the direct-transfer mechanism and inconsistent with a mechanism in which free NADH is the only competent substrate for either enzyme-catalyzed reaction. The discrepancy between this conclusion and that of Chock and Gutfreund comes from (i) their incorrect measurement of the K. for NADH in the a-GDH-catalyzed reaction, (a) inadequate design and range of the steady-state kinetic experiments, and (ii) their qualitative assessment of the prediction of the direct-transfer mechanism.Our transient kinetic measurements for the transfer of NADH from a-GDH to LDH and from LDH to a-GDH show that both are slower than predicted on the basis of free equilibration of NADH through the aqueous environment. The decrease in the rate of equilibration of NADH between a-GDH and LDH provides no support for the random-diffusion mechanism; rather, it suggests a direct interaction between enzymes that modulates the transfer rate of NADH. Thus, contrary to Chock and Gutfreund's conclusion, all our experimental data compel us to propose, once again, that NADH is transferred directly between the sites of a-GDH and LDH.In a series of communications over the past 6 years, we have reported that an enzyme-bound metabolite can often serve as a substrate for another enzyme-catalyzed reaction (1, 2). This view is in contrast to the widely held hypothesis that, except for intermediary steps in multienzyme complexes, the substrates and products of enzyme-catalyzed reactions are bound directly from and released directly into the aqueous solution (3). However, since the concentrations of enzymes often exceed the concentrations of their affine metabolites under physiological conditions, the universal application of a free-diffusion mechanism is not clear, and it is a justifiable subject for detailed experimental scrutiny (1, 2). Toward this end, we have distinguished the two extremes of mechanism of metabolite transfer between enzyme pairs that share a common metabolite (Eq. 1):In the random-diffusion mechanism, the enzyme-bound metabolite first dissociates into the aqueous solvent and then binds at the second enzyme site, wh...

Data from steady-state kinetic analysis of yeast K+-activated aldehyde dehydrogenase are consistent with a ternary complex mechanism. Evidence from alternative substrate analysis and product-inhibition studies supports an ordered sequence of substrate binding in which NAD+ is the leading substrate. A preincubation requirement for NAD+ for maximum activity is also consistent with the importance of a binary enzyme-NAD+ complex. Dissociation constant for enzyme-NAD+ complex determined kinetically is in reasonable agreement with that determined by direct binding. The order of substrate addition proposed here differs from that proposed for a yeast aldehyde dehydrogenase previously reported. Different methods of purification produced an enzyme that showed similar kinetic characteristics to those reported here.

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A convenient and highly specific continuous spectrophotometric assay for sodium-potassium adenosine triphosphate activity utilizing the rapidly hydrolyzed and high-affinity chromophoric substrate beta-(2-furyl)acryloyl phosphate (FAP) is described. The Na/K-ATPase-catalyzed hydrolysis of FAP is faster than that for ATP under all ionic conditions. The rate is neither inhibited nor activated by Na+; it is dependent on [K+] and on [Mg2+]. The hydrolysis of FAP to furylacrylate is accompanied by a large shift in the UV absorbance maximum. The spectrum of FAP, but not furylacrylate, is sensitive to noncovalent ligation with Mg2+, a happenstance which permits the identification of Mg2+FAP, and consequently allows for a probe of the role of Mg2+ in the catalysis. Mg2+ binding to the active site is essential for catalysis. MgFAP is more tightly bound to the site than is FAP2-, but the complex is not obligatory for catalysis. The formation of a phosphoryl-enzyme intermediate is not evident in the reaction of FAP with the enzyme. Transient kinetic experiments, utilizing an excess of MgFAP, demonstrate a unique steady-state rate-limiting production of furylacrylate. These results indicate that the pathway demonstrated with ATP is not appropriate to the FAPase mechanism. The results suggest that acyl phosphates are good "phosphatase" substrates either because they are analogues of the phosphatase-specific phosphoryl-enzyme or because they react exclusively with the isomerized "E2" form of the enzyme.