Stereospecificity of C4 nicotinamide hydrogen transfer of the NADP-dependent glyceraldehyde-3-phosphate dehydrogenase.

The stereospecificity of the reaction catalysed by the spinach chloroplast enzyme NADP-dependent glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating), EC 1.2.1.13) with respect to the C4 nicotinamide hydrogen transfer was investigated. NADPH deuterated at the C4 HA position was synthesized using aldehyde dehydrogenase. 1H-NMR spectroscopy was used to examine the NADP+ product of the GPDH reaction for the presence or absence of the C4 deuterium atom. Chloroplast NADP-dependent glyceraldehyde-3-phosphate dehydrogenase retains the deuterium at the C4 HA position (removing the hydrogen atom), and is therefore a B (pro-S) specific dehydrogenase.

Interaction of NADPH and triazine dyes with ferredoxin-NADP+ oxidoreductase.

The ability of NADPH to compete for binding with other ligands of known affinity has been used to provide values for the Kd of NADPH with ferredoxin-NADP+ oxidoreductase (EC 1.18.1.2) (FNR). When the competing ligand is procion red, which binds with a red-shift in spectrum, or Woodwards reagent K(N-ethyl-5-phenylisoxazolium 3'-sulfonate), which covalently modifies an active site carboxyl residue, the calculated Kd for the NADPH-FNR complex is greater than 8 or 0.08 mM, respectively. Because of the feeble (or non-existent) ability of NADPH to dislodge procion red, we propose that this dye and NADPH are not binding at the same site. Procion red must, however, bind additionally at the active site (presumably without spectral perturbation) as it is a competitive inhibitor of NADPH in ferricyanide reduction assays and more crucially proves to be a novel substrate itself, being reduced to a leuco form which can be reoxidised by oxygen. Although a Kd for the NADPH-FNR complex of 0.08 mM is reasonable, we point out the difficulty of interpreting this value and question its physiological significance.

Double mixing technique for linked enzyme assays.

A rapid double mixing technique has been applied to the reaction kinetics of enzymes usually analyzed by linked assay. The test enzyme is allowed to perform in the absence of the linking enzyme but then the convenience of enzymatic analysis of accumulated produce is exploited when a second mixing adds the linking enzyme. The analytical method is presented which enables the determination of the rate of product accumulation during the delay between mixings. Various advantages of the system in both steady-state and transient kinetic analyses are described. As an example, 3-phosphoglycerate kinase is assayed. The results address the proposal that the kinase interacts with the usual linking enzyme, glyceraldehyde-3-phosphate dehydrogenase, and also raise questions about product inhibition.

The rationalization of high enzyme concentration in metabolic pathways such as glycolysis.

The cellular concentration of enzymes of some major metabolic pathways, such as glycolysis, can approach millimolarity. This concentration of enzyme can catalyze in vitro rates which are 100-fold higher than maximum pathway flux. In an attempt to understand the need for such high enzyme concentration, an artificial metabolic pathway of five enzymes (apropos the central enzymes of glycolysis) has been modeled. Numerical methods were then used to determine the effect of enzyme concentration on: (1) the change in total free metabolite concentration as the pathway changes from low flux to high flux, (2) the time lag (transient time) in the rate of final product formation upon the transition from low flux to high flux. Both the changes in metabolite pool size and the transient time decreased with increased enzyme concentrations. When all enzymatic reactions were assigned Keq of unity, a concentration for each enzyme of 25 microM is sufficient to provide a transient time of 1 sec. When Keq different from unity are introduced, more enzyme is required to provide comparably short transient times. Under the latter condition, a pathway of sufficiently low transient time would require all the enzyme available in mammalian muscle. It is shown that there is little scope for further increases in either enzyme concentration or of catalytic efficiency of independent enzymes. Therefore, an alternative method of increasing efficiency is considered in which enzyme-bound metabolites can serve directly as substrates for subsequent enzymes in a metabolic pathway.

Kinetics and reaction mechanism of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

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.

Rapid purification and properties of potassium-activated aldehyde dehydrogenase from Saccharomyces cerevisiae.

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.

Multiple binding of thallium and rubidium to potassium-activated yeast aldehyde dehydrogenase. Influences on tertiary structure, stability and catalytic activity.

Univalent cation activators of aldehyde dehydrogenase have dual effects, both interpreted as cation-induced or -stabilized conformation changes. These two processes are differentiated by the time scales of their associated changes in activity. Using Tl+ as an activator, under certain conditions, the slower change in activity saturates at a Tl+ concentration which is only 0.1 Ks for the faster change. This, together with evidence for cation-induced rather than cation-stabilized conformation changes, is used to propose separate binding sites for cations responsible for the two activation processes. Equilibrium dialysis indicates 4 binding sites per active site for Rb+ or 6 sites for Tl+. At least one of the additional sites for Tl+ is an inhibitory site which has been differentiated from the activator sites on the basis of steady-state and pre-steady-state kinetic data.

Kinetic and spectroscopic evidence of cation-induced conformation changes in yeast K+ -activated aldehyde dehydrogenase.

The activity, stability and spectroscopic properties of yeast K+ -activated aldehyde dehydrogenase were measured at various times after removal from, and after returning to a solution containing K+. Enzyme activity is rapidly lost on removal of most of the K+ and rapidly regained if K+ is replaced immediately. These activity changes are slower than likely rates of K+ dissociation and association. These rapid changes in concentration result in altered enzyme stability with enzyme in K+ the more stable. U.v. difference spectra are produced whenever enzyme in an activating environment (K+ or Tl+) is compared with enzyme in a non-activating environment (Tris+ or Li+). These spectral changes occur within 10s. The saturation characteristics with K+ are hyperbolic for all three phenomena of activation, stabilization and spectral change, with estimated apparent dissociation constants (Ks) for K+ of 7.5 mM, 5.5 mM and 6 mM respectively. Continued incubation of enzyme in the absence of K+ results in the accumulation of an enzyme form that re-activates only slowly on replacing K+. Stability characteristics in various concentrations of K+ over equivalent time scales are consistent with the existence of additional conformations. Spectroscopic evidence also indicates such additional slow conformation changes. Results have been interpreted in terms of two separate conformation transitions induced or stabilized by K+.

Direct transfer of NADH between alpha-glycerol phosphate dehydrogenase and lactate dehydrogenase: fact or misinterpretation?

Following the criticism by Chock and Gutfreund [Chock, P.B. & Gutfreund, H. (1988) Proc. Natl. Acad. Sci. USA 85, 8870-8874], that our proposal of direct transfer of NADH between glycerol-3-phosphate dehydrogenase (alpha-glycerol phosphate dehydrogenase, alpha-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, 4538-4545], we examined the mechanism (random diffusion vs. direct transfer) of transfer of NADH between rabbit muscle alpha-GDH and pig heart LDH. The steady-state data reveal that the LDH-NADH complex and the alpha-GDH-NADH complex can serve as substrate for the alpha-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 Km for NADH in the alpha-GDH-catalyzed reaction, (ii) inadequate design and range of the steady-state kinetic experiments, and (iii) their qualitative assessment of the prediction of the direct-transfer mechanism. Our transient kinetic measurements for the transfer of NADH from alpha-GDH to LDH and from LDH to alpha-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 alpha-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 alpha-GDH and LDH.