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D B Northrop

Publications and source records attributed to D B Northrop.

At least 19 recordsLinked to original sources
PubMed2001

Effect of pressure on deuterium isotope effects of formate dehydrogenase.

High pressure causes biphasic effects on the oxidation of formate by yeast formate dehydrogenase as expressed on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by accelerating hydride transfer. The transition state for hydride transfer has a smaller volume than the free formate plus the capturing form of enzyme, with DeltaV(double dagger) = -9.7 +/- 1.0 mL/mol. Pressures above 1.5 kbar decrease capture, reminiscent of effects on the conformational change associated with the binding of nicotinamide adenine dinucleotide (NAD(+)) to yeast alcohol dehydrogenase [Northrop, D. B., and Y. K. Cho (2000) Biochemistry 39, 2406-2412]. The collision complex, E-NAD(+), has a smaller volume than the more tightly bound reactant-state complex, E-NAD(+), with DeltaV = +83.4 +/- 5.2 mL/mol. A comparison of the effects of pressure on the oxidation of normal and deuteroformate shows that the entire isotope effect on hydride transfer, 2.73 +/- 0.20, arises solely from transition-state phenomena, as was also observed previously with yeast alcohol dehydrogense. In contrast, normal primary isotope effects arise solely from different zero-point energies in reactant states, and those that express hydrogen tunneling arise from a mixture of both reactant-state and transition-state phenomena. Moreover, pressure increases the primary intrinsic deuterium isotope effect, the opposite of what was observed with yeast alcohol dehydrogense. The lack of a decrease in the isotope effect is also contrary to empirical precedents from chemical reactions suspected of tunneling and to theoretical constructs of vibrationally enhanced tunneling in enzymatic reactions. Hence, this new experimental design penetrates transition states of enzymatic catalysis as never before, reveals the presence of phenomena foreign to chemical kinetics, and calls for explanations of how enzymes work beyond the tenants of physical organic chemistry.

Candida↗
PubMed2001

Uses of isotope effects in the study of enzymes.

There have been few recent additions to the technical methods employed in the study of isotope effects, notable exceptions being the use of high pressure as an experimental variable and the measurement of heavy-atom isotope effects on maximal velocities using continuous-flow techniques. Most of the innovations are in the realm of new experimental designs that allow the asking of new questions. These designs include the use of isotope effects to: determine kinetic mechanisms, distinguish between changes in enzymatic activity and loss of active enzyme, distinguish between reactant-state origins and transition-state origins and quantify hydrogen tunneling, separate and quantify multiple origins of solvent isotope effects, distinguish between concerted and stepwise chemical mechanisms, characterize bond order changes in ligand binding, distinguish different pathways of inhibitor binding, and estimate intrinsic isotope effects.

Biochemistry↗
PubMed2001

Follow the protons: a low-barrier hydrogen bond unifies the mechanisms of the aspartic proteases.

Seven proton transfers in five steps participate in a catalytic turnover of an aspartic protease. The Rosetta Stone for elucidating their role is a low-barrier hydrogen bond that holds the two aspartic carboxyls in a coplanar conformation. The proton of this bond shuttles between oxygens during chemical steps via hydrogen tunneling, unlike in previous proposals where it was transferred to substrate. After the release of products, both carboxyls are protonated and the bond is missing. Re-forming the bond is a significant step within a kinetic isomechanism. The bond also explains-at long last-the extremely low pK in pH profiles.

Aspartic Acid Endopeptidases↗
PubMed2000

Effect of pressure on deuterium isotope effects of yeast alcohol dehydrogenase: evidence for mechanical models of catalysis.

Moderate pressure accelerates hydride transfer catalyzed by yeast alcohol dehydrogenase, indicative of a large negative volume of activation [Cho and Northrop (1999) Biochemistry 38, 7470-7475]. A comparison of the effects of pressure on the oxidation of normal versus dideuteriobenzyl alcohol generates a monophasic decrease in the intrinsic isotope effect; therefore, the volume of activation for the transition-state of deuteride transfer must be even more negative, by 10.4 mL/mol. This finding appears consistent with hydrogen tunneling previously proposed for this dehydrogenase [Cha, Y., Murray, C. J., and Klinman, J. P. (1989) Science 243, 1325-1330]. However, a global fit of the primary data shows that the entire isotope effect arises from a transition-state phenomenon, unlike normal isotope effects, which arise from different vibrational frequencies in reactant states, and tunneling isotope effects, which arise from a mixture of both states. Assuming the phenomenon is tunneling, the isotopic data are consistent with a Bell tunneling correction factor of Q(H) = 12 and an imaginary frequency of nu(H) = 1220 cm(-1), the first so calculated from experimental enzymatic data. This excessively large correction factor and the large difference in the isotopic activation volumes, plus the low isotope effects at extrapolated pressures, challenge traditional applications of physical organic chemistry and transition-state theory to enzymatic catalysis. They suggest instead that something other than transition-state stabilization or tunneling is responsible for the rate acceleration, something unique to the enzymatic transition state that does not occur in nonenzymatic reactions. Arguments for the vibrational model of coupled atomic motions and the fluctuating enzyme model of protein domain motion are put forward as possible interpretations.

Alcohol Dehydrogenase↗
PubMed2000

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.

Alcohol Dehydrogenase↗
PubMed1999

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 DeltaV 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 DeltaV of 73 +/- 2 mL/mol. The equilibrium constant of the conformational change Keq is 75 +/- 13 at 1 atm. The effects of pressure on the capture of NAD+ have no activation phase because the conformational change is now being expressed kinetically instead of thermodynamically, together with but in opposition to hydride transfer, causing the effects to cancel. For yeast alcohol dehydrogenase, this conformational change had not been detected previously, but similar conformational changes have been found by spectroscopic means in other dehydrogenases, and some of them are also sensitive to pressure. The opposite signs for the volume change of tighter binding and hydride transfer run contrary to Pauling's hypothesis that substrates are bound more tightly in the transition state than in the Michaelian reactant state.

Alcohol Dehydrogenase↗
PubMed1999

Rethinking fundamentals of enzyme action.

Despite certain limitations, investigators continue to gainfully employ concepts rooted in steady-state kinetics in efforts to draw mechanistically relevant inferences about enzyme catalysis. By reconsidering steady-state enzyme kinetic behavior, this review develops ideas that allow one to arrive at the following new definitions: (a) V/K, the ratio of the maximal initial velocity divided by the Michaelis-Menten constant, is the apparent rate constant for the capture of substrate into enzyme complexes that are destined to yield product(s) at some later point in time; (b) the maximal velocity V is the apparent rate constant for the release of substrate from captured complexes in the form of free product(s); and (c) the Michaelis-Menten constant K is the ratio of the apparent rate constants for release and capture. The physiologic significance of V/K is also explored to illuminate aspects of antibiotic resistance, the concept of "perfection" in enzyme catalysis, and catalytic proficiency. The conceptual basis of congruent thermodynamic cycles is also considered in an attempt to achieve an unambiguous way for comparing an enzyme-catalyzed reaction with its uncatalyzed reference reaction. Such efforts promise a deeper understanding of the origins of catalytic power, as it relates to stabilization of the reactant ground state, stabilization of the transition state, and reciprocal stabilizations of ground and transition states.

Anti-Bacterial Agents↗
PubMed1998

Transpeptidation by porcine pepsin catalyzed by a noncovalent intermediate unique to its iso-mechanism.

Porcine pepsin proteolysis of the hexapeptide Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe (where OMe = methoxy and Nle = norleucine) in the presence of dipeptide Leu-Leu synthesizes a new hexapeptide Leu-Ser-p-nitro-Phe-Leu-Leu. Contrary to transpeptidation kinetics of other proteases, which depend upon an acyl-enzyme intermediate, the time course for pepsin-catalyzed transpeptidation displays a distinct lag before reaching a steady-state reaction velocity. Moreover, this lag is coupled to burst kinetics for the formation of proteolytic products, Leu-Ser-p-nitro-Phe and Nle-Ala-Leu-OMe. The lag requires that free Leu-Ser-p-nitro-Phe accumulate in the reaction medium during the lag phase and subsequently rebind for transpeptidation. Consistent with this dissociative kinetic mechanism are normal solvent isotope effects on formation of the proteolytic products Leu-Ser-p-nitro-Phe (vH/vD = 2.2 +/- 0.2) and Nle-Ala-Leu-OMe (vH/vD = 1.8 +/- 0.1) as opposed to an inverse effect on the formation of the transpeptidation product Leu-Ser-p-nitro-Phe-Leu-Leu (vH/vD = 0.40 +/- 0.09). Because proteolysis is slower in D2O but transpeptidation is faster, the isotopically sensitive step must occur after release of both products of proteolysis, which precludes putative acyl-enzyme covalent intermediates. Isotopically enhanced transpeptidation is a new type of isotope effect but one that is consistent with the Uni Bi iso-mechanism previously postulated on the basis of solvent isotope effects on Vmax but not on Vmax/Km (Rebholz, K. L., and Northrop, D. B. (1991) Biochem. Biophys Res. Commun. 179, 65-69) and confirmed by solvent isotope effects on the onset of inhibition by pepstatin (Cho, Y.-K., Rebholz, K. L., and Northrop, D. B. (1994) Biochemistry 33, 9637-9642). As a new biochemical mechanism for peptide bond synthesis that has a potential for applications in biotechnology, it is here proposed that the energy necessary to drive peptide synthesis from free peptides comes from the sizable free energy drop associated with rehydration of the active site of pepsin in 55 M water.

Animals↗
PubMed1998

Kinetics of enzymes with isomechanisms: britton induced transport catalyzed by bovine carbonic anhydrase II, measured by rapid-flow mass spectrometry.

Induced transport of 13CO2 to H13CO3- by bovine carbonic anhydrase II in the presence of excess H12CO3- at pH 6.35 causes a temporary decrease in the concentration of 13CO2 from 0.169 to 0.092 +/- 0.003 mM, measured in less than half a second with a new rapid-flow, membrane-inlet mass spectrometer. From this perturbation, a value of 83 +/- 0.3 M-1 is calculated for the alpha term in Eq. [26] of H. G. Britton (Biochem. J. 133, 255-261, 1973). Combining alpha with the K(m) for bicarbonate (32 +/- 1 mM) and Eqs. [7] and [21] of K. L. Rebholz and D. B. Northrop (Methods Enzymol, 249, 211-240, 1995) yields a ratio of less than 0.57 +/- 0.04 for the apparent rate constants representing the isomerization segment and chemical conversion segment, respectively, of the enzyme-catalyzed dehydration of bicarbonate. These results provide proof positive for the previously inferred (but unproven despite universal acceptance) isomechanism for the carbonic anhydrases. Moreover, the data and new equations quantify the proposed internal proton transfer to be 64 +/- 4% rate-limiting for the bovine type II isoenzyme, a value similar to but more precise than estimates based upon solvent isotope effects and product inhibition kinetics.

Animals↗
PubMed1997

Kinetics of enzymes with iso-mechanisms: solvent isotope effects.

Kinetic isotope effects on enzymatic reactions which employ general acid or general base catalytic mechanisms may arise during reprotonations of free enzyme. These effects reveal kinetically significant isomerizations of the free enzyme, or iso-mechanisms. The effects are expressed kinetically at high concentrations of substrate, on Vmax or Kcat, but only thermodynamically at low substrate, on Vmax/K(m). The effects are also expressed on the noncompetitive inhibition constant of product inhibition, Kiip, because this parameter is dependent upon the steady-state concentration of the product form of free enzyme. A normal isotope effect on isomerization will decrease Vmax and Kiip, but not necessarily to the same degree. Which is greater will depend upon how rate-limiting the isomerization is to a complete turnover. Together they are related to the full effect on isomerization, DKiso, by their product: DKiso = DVmax DKiip. Moreover, precisely how rate-limiting the isomerization is to a turnover can be shown to be numerically equal to (DVmax - 1)/(DKiipDVmax - 1), which surprisingly, holds whether there are other isotope effects present or not. The new relationships applied to published data on bovine carbonic anhydrase II reveal an intrinsic solvent isotope effect of DK = 9 +/- 4, and an iso step that is less than 80% rate-limiting. Applied to porcine pepsin, a significant DV is accompanied by excessive standard error on DKiip, precluding the calculation of a definitive intrinsic solvent isotope effect.

Carbonic Anhydrases↗
PubMed1997

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.

Catalysis↗
PubMed1994

Solvent isotope effects on the onset of inhibition of porcine pepsin by pepstatin.

Pepstatin is a slow and tight-binding inhibitor of pepsin. Preincubating enzyme and inhibitor in H2O and in D2O in the absence of substrate generates an inverse solvent isotope effect of Dk = 0.69 +/- 0.06 on the apparent first-order rate constant for the decay in enzymatic activity. Proton inventory analysis of the inverse isotope effect suggests a single transition-state proton with a fractionation factor of 1.41 +/- 0.05. In contrast, combining enzyme with inhibitor and substrate (Leu-Ser-p-nitro-Phe-Nle-Ala-Leu-OMe) simultaneously along with observing the decay in enzymatic activity during catalytic turnovers generates a normal solvent isotope effect of Dk = 1.25 +/- 0.09. Proton inventory analysis of the normal isotope effect suggests a single reactant-state proton with a fractionation factor of 1.46 +/- 0.03. These two experimental designs are often considered equivalent, but the differences in isotopic data require that the pathway for onset of pepstatin inhibition in the absence of substrate must be different from the pathway in the presence of substrate. In the former, the inhibitor can only bind to free enzyme; in the latter, the inhibitor is hindered from binding to free enzyme because of competition with substrate but can bind to intermediate forms of enzyme generated during catalytic turnovers, downstream from enzyme-product complexes.

Animals↗
PubMed1994

Kinetics of enzymes with iso-mechanisms: analysis and display of progress curves.

Isomerizations of free enzyme can be detected in progress curves as a deviation from linearity when plotted according to the linear transformation of integrated rate equations of Foster and Niemann. Iso-mechanisms can also be detected as a second inhibition constant when sets of progress curves are fitted by nonlinear regression to the integrated form of appropriate rate equations. The latter is extremely sensitive and can detect the presence of the additional inhibition constant from relationships between progress curves, even when a deviation from linearity is not apparent within individual plots using the graphical method. Both methods can detect iso-mechanisms from data that do not express oversaturation kinetics.

Binding, Competitive↗
PubMed1994

Kinetics of enzymes with iso-mechanisms: dead-end inhibition of fumarase and carbonic anhydrase II.

Isomerization of free enzyme can be detected in kinetic patterns of dead-end inhibition because competitive substrate analogs yield noncompetitive inhibition versus product in reverse reaction kinetics. The ratio of slope and intercept inhibition constants allows a quantitative estimation of the relative kinetic significance of the isomerization to a catalytic turnover. Applying this kinetic analysis theoretically to inhibition data for bovine carbonic anhydrase II by anions [Y. Pocker and T. L. Deits (1982) J. Am. Chem. Soc. 104, 2424] provides an estimate of 43 +/- 13% for how rate-limiting the isomerization segment is at pH 6.6. Applying the analysis experimentally to porcine heart fumarase provides a competitive pattern of inhibition by trans-aconitate versus fumarate with Ki(s) = 2.0 +/- 0.5 mM, together with a non-competitive pattern versus malate, with Ki(s) = 0.8 +/- 0.1 mM and Kii = 2.3 +/- 0.4 mM. Assuming that the isomerization segment of fumarase is the reprotonation of an active site carboxyl and imidazole with pK1 = 5.53 and pK2 = 7.78 [Blanchard and Cleland (1980) Biochemistry 19, 4506], an apparent rate constant for the isomerization segment of fumarate hydration is estimated as 95 +/- 22 s-1, compared to 42 +/- 13 s-1 for the chemical segment and 29 +/- 0.7 s-1 for a complete turnover. In contrast, the values are 17000 +/- 5200, 82 +/- 25, and 82 +/- 3 s-1, respectively, for malate dehydration. Hence, the isomerization segment is 30 +/- 7% rate-limiting during fumarate hydration but less than 1% during malate dehydration.

Aconitic Acid↗
PubMed1993

Kinetics of enzymes with iso-mechanisms: analysis of product inhibition.

Isomerizations of free enzyme can be detected in kinetic patterns of product inhibition when the isomerization is partially rate-limiting. The kinetic pattern is non-competitive, owing to binding of substrate and product to different forms of free enzyme. This adds an additional term to the rate equation, sometimes represented as KSP. Several kineticists have noted that, as the rate of isomerization becomes high in relation to catalytic turnover, the intercept effect will become small, KSP will approach infinity, and the pattern will look competitive. Britton [(1973) Biochem. J. 133, 255-261] asserted that KSP will also approach infinity when the rate of isomerization becomes low. This second assertion is incorrect and can be traced to the particular model and graphical representation used to examine KSP as a function of relative rate constants. The function portrayed as a parabola with two roots for KSP is, instead, a straight line with one root. The algebraic condition justifying the second root obtains in the limit of zero in the rate of reaction and thus is not experimentally relevant, and the appearance of competitive inhibition, based on KSP alone, is not valid. Using a more general model, new equations are derived and presented which provide direct calculations of the apparent rate constants for free enzyme isomerizations from product-inhibition data when the equilibrium of the isomerization is near 1, and useful limits for the rate constants when greater than or less than 1.

Binding, Competitive↗