Dexter B. Northrop

[30] Deuterium and tritium kinetic isotope effects on initial rates

Publisher Summary An enzyme-catalyzed reaction may display a very broad range of values for kinetic isotope effects, depending on reaction conditions. Because the methods of determination and ways of thinking about isotope effects originate with chemical reactions, the variation of values has led to considerable conceptual difficulty, poorly designed experiments, and faulty interpretation of results. Thus, this chapter defines the variety of hydrogen isotope effects within a logical and practical framework, and outlines appropriate experimental designs for the determination of each. It is reported that the primary limitation encountered in the use of isotope effects in enzymology is the need for extreme precision. Unlike steady-state kinetics, which are concerned with parameter estimations, isotope effects are defined as ratios of parameters that, in addition, have quantitative significance only as diminished ratios to yet another isotope effect.

New concepts in bioorganic chemistry beyond enzyme kinetics: Direct determination of mechanisms by stopped-flow mass spectrometry

The development of soft ionization techniques has made mass spectrometry an efficient and essential tool for the determinations of the primary structures of peptides and proteins. Recently the technique has been extended at an explosive rate to noncovalent structures as well as dynamics of protein-protein interactions. We propose here that interfacing mass spectrometry with a stopped-flow mixing device and applying these new techniques of soft ionization to enzymes undergoing catalysis will provide direct access to enzyme mechanisms, both kinetic mechanisms (which describe the comings and goings of substrates, products, and inhibitors) and chemical mechanisms (which describe the order of breaking and making chemical bonds). Transient-state measurements will provide the order of reaction events; steady-state measurements will provide the distribution and therefore the relative energy level of enzyme forms participating in those events; combining transient-state and steady-state measurements is therefore expected to provide sufficient information to construct a free energy diagram of the enzyme-catalyzed reaction.

Fitting enzyme-kinetic data to VK

Kinetic data from enzyme-catalyzed reactions have been analyzed traditionally in terms of the Michaelis-Menten equation, which assumes that the maximal velocity (V) and the Michaelis constant (K) are the primary kinetic constants. But what is needed from most kinetic studies today is V/K. A new form of the equation is proposed which assumes that V and V/K are the primary kinetic constants: v = (V . S . V/K)/(V + S . V/K). Computer fittings of both experimental and simulated velocity data to both equations give results favoring the new equation.

[36] Oxaloacetate transcarboxylase from Propionibacterium☆

Publisher Summary This chapter deals with the Oxaloacetate transcarboxylase from propionibacterium. The enzyme activity is assayed spectrophotometrically by determining oxaloacetate formation through coupling with malate dehydrogenase. This assay is used routinely, except when lactate dehydrogenase or DPNH oxidase is present. If the latter are present in small amounts, a control value is determined by omission of methylmalonyl-CoA and is subtracted from the value obtained with the complete assay mixture. When these contaminants are excessive, the reaction is carried out without the addition of malate dehydrogenase and DPNH, and after a suitable interval the mixture is deproteinized with trichloroacetic acid and the oxaloacetate is determined in the neutralized solution using malate dehydrogenase. The direct assay with correction for the control usually can be used for the purification described below and thereafter the control value is so small that it may be neglected. Transcarboxylase is isolated from the bacteria grown in lactate, glucose, or glycerol. The yields of cells and transcarboxylase are quite similar from glucose or glycerol media; glycerol has the advantage that it does not cause caramelization when sterilized with other constituents of the medium, and contamination by other bacteria is less likely with this substrate.

Effects of pressure on the kinetics of capture by yeast alcohol dehydrogenase.

High pressure causes biphasic effects on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase as expressed in the kinetic parameter V/K which measures substrate capture. Moderate pressure increases the rate of capture of benzyl alcohol by activating the hydride transfer step. This means that the transition state for hydride transfer has a smaller volume than the free alcohol plus the capturing form of enzyme, with a ΔV⧧ of −39 ± 1 mL/mol, a value that is relatively large. This is the first physical property of an enzymatic transition state thus characterized, and it offers new possibilities for structure−activity analyses. Pressures of >1.5 kbar decrease the rate of capture of benzyl alcohol by favoring a conformation of the enzyme which binds nicotinamide adenine dinucleotide (NAD+) less tightly. This means that the ground state for tight binding, E*−NAD+, has a larger volume than the collision complex, E−NAD+, with a ΔV* of 73 ± 2 mL/mol. The equilibrium constant of the conformational change K...

Unusual origins of isotope effects in enzyme-catalysed reactions

High hydrostatic pressure is a neglected tool for probing the origins of isotope effects. In chemical reactions, normal primary deuterium isotope effects (DIEs) arising solely from differences in zero point energies are unaffected by pressure; but some anomalous isotope effects in which hydrogen tunnelling is suspected are partially suppressed. In some enzymatic reactions, high pressure completely suppresses the DIE. We have now measured the effects of high pressure on the parallel 13C heavy atom isotope effect of yeast alcohol dehydrogenase and found that it is also suppressed by high pressure and, similarly, suppressed in its entirety. Moreover, the volume changes associated with the suppression of both deuterium and heavy atom isotope effects are virtually identical. The equivalent decrease in activation volumes for hydride transfer, when one mass unit is added to the carbon end of a scissile C–H bond as when one mass unit is added to the hydrogen end, suggests a common origin. Given that carbon is highly unlikely to undergo tunnelling, it follows that hydrogen is not doing so either. The origin of these isotope effects must lie elsewhere. We offer protein domain motions as a possibility.

Effects of High Pressure on Solvent Isotope Effects of Yeast Alcohol Dehydrogenase

The effect of pressure on the capture of a substrate alcohol by yeast alcohol dehydrogenase is biphasic. Solvent isotope effects accompany both phases and are expressed differently at different pressures. These differences allow the extraction of an inverse intrinsic kinetic solvent isotope effect of 1.1 (i.e., (D(2(O)))V/K = 0.9) accompanying hydride transfer and an inverse equilibrium solvent isotope effect of 2.6 (i.e., (D(2(O)))K(s) = 0.4) accompanying the binding of nucleotide, NAD(+). The value of the kinetic effect is consistent with a reactant-state E-NAD(+)-Zn-OH(2) having a fractionation factor of phi approximately 0.5 for the zinc-bound water in conjunction with a transition-state proton exiting a low-barrier hydrogen bond with a fractionation factor between 0.6 and 0.9. The value of the equilibrium effect is consistent with restrictions of torsional motions of multiple hydrogens of the enzyme protein during the conformational change that accompanies the binding of NAD(+). The absence of significant commitments to catalysis accompanying the kinetic solvent isotope effect means that this portion of the proton transfer occurs in the same reactive step as hydride transfer in a concerted chemical mechanism. The success of this analysis suggests that future measurements of solvent isotope effects as a function of pressure, in the presence of moderate commitments to catalysis, may yield precise estimates of intrinsic solvent isotope effects that are not fully expressed on capture at atmospheric pressure.

Purification and spectrophotometric assay of neomycin phosphotransferase II1

Summary Neomycin phosphotronnferase II is maximally released by osmatic shocking of R+ E. coli between late log and early stationary phase. A 300–400-fold purification of the enzyme protein is accomplished by streptomycin sulfate and ammonium sulfate precipitations of osmotic shockates, followed by affinity and ion-exchange chromatography. The recovered enzyme preparation is electrophoretically 90% pure, is free of ATP-ase activity, and can be conveniently assayed spectrophotometrically by linking the production of ADP to pyruvate kinase and lactate dehydrogenase. The purified enzyme, however, is not stable.

[9] Kinetics of iso mechanisms

Publisher Summary Methods for the detection and analysis of enzymatic Iso mechanisms have been simplified and expanded in this chapter. The simplification is not the result of simplifying assumptions, but rather of a conversion of kinetic terms into standard nomenclature, together with the derivation of new equations. The expansion includes: (a) new methods to fit progress curves to detect and quantify noncompetitive product inhibition, (b) a second method to detect α iso based on noncompetitive dead-end inhibitors, and (c) an analysis of the expression of kinetic isotope effects, from within an Iso segment, together with a means to calculate the intrinsic isotope effect. With less intimidating algebra than offered previously, enzymologists should find the consideration of an Iso mechanism more inviting and, inevitably, more enzymes will be found to possess Iso mechanisms. The results to date show that the addition and release of water and protons from an enzyme active site need not be in rapid equilibrium as generally been assumed. This finding alone necessitates the reanalysis of published data for enzymes, in which the movement of protons or water are involved, and this projected analysis most likely will produce many candidates for further study.

Slow step after bond-breaking by porcine pepsin identified using solvent deuterium isotope effects

The relatively fast artificial substrate Leu-Ser-rho-nitro-Phe-Nle-Ala-Leu-OMe generates a solvent isotope effect of 1.51 +/- 0.02 only on the maximal velocity of peptide hydrolysis catalyzed by porcine pepsin (EC 3.4.23.1). The absence of an isotope effect on V/K places the isotopically-sensitive step after peptide bond cleavage and the release of the first product. Reprotonation of the active site aspartic carboxyls is proposed as the most likely interpretation of this observation. Structural and kinetic similarities between pepsin and other aspartic proteinases, including the therapeutically important targets HIV protease and renin, suggest a similar slow reprotonation step after catalysis. This mechanistic feature has important implications regarding inhibitor design; if most of the enzymes are present in a product-release form during steady-state turnover, then perhaps inhibitors should be designed as product analogs instead of substrate analogs.