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• W2031569498 endingPage "51984" @default.
• W2031569498 startingPage "51975" @default.
• W2031569498 abstract "When our laboratory started to carry out kinetic experiments on enzyme-catalyzed reactions we focused originally on initial velocity studies in which the concentrations of substrates, products, and inhibitors were varied. The notation and theory for these types of experiments were published as three papers in Biochimica et Biophysica Acta that have received many citations over the years (1Cleland W.W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. I. Nomenclature and rate equations..Biochim. Biophys. Acta. 1963; 67: 104-137Crossref PubMed Google Scholar, 2Cleland W.W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. II. Inhibition: nomenclature and theory..Biochim. Biophys. Acta. 1963; 67: 173-187Crossref PubMed Google Scholar, 3Cleland W.W. The kinetics of enzyme-catalyzed reactions with two or more substrates or products. III. Prediction of initial velocity and inhibition patterns by inspection..Biochim. Biophys. Acta. 1963; 67: 188-196Crossref PubMed Google Scholar). We used these methods to study various enzymes over the next decade or so. Although we used isotopes to measure isotopic exchanges in creatine kinase (4Morrison J.F. Cleland W.W. Isotope exchange studies of the mechanism of the reaction catalyzed by adenosine triphosphate: creatine phosphotransferase..J. Biol. Chem. 1966; 241: 673-683Abstract Full Text PDF PubMed Google Scholar), galactokinase (5Gulbinsky J.S. Cleland W.W. Kinetic studies of Escherichia coli galactokinase.Biochemistry. 1968; 7: 566-575Crossref PubMed Scopus (63) Google Scholar), shikimate dehydrogenase (6Balinsky D. Dennis A.W. Cleland W.W. Kinetic and isotope-exchange studies on shikimate dehydrogenase from Pisum sativum..Biochemistry. 1971; 10: 1947-1952Crossref PubMed Scopus (29) Google Scholar), alcohol dehydrogenase (7Ainslie Jr., G.R. Cleland W.W. Isotope exchange studies on liver alcohol dehydrogenase with cyclohexanol and cyclohexanone as reactants..J. Biol. Chem. 1972; 247: 946-951Abstract Full Text PDF PubMed Google Scholar), and isocitrate dehydrogenase (8Uhr M.L. Thompson V.W. Cleland W.W. The kinetics of pig heart triphosphopyridine nucleotide-isocitrate dehydrogenase. I. Initial velocity, substrate and product inhibition, and isotope exchange..J. Biol. Chem. 1974; 249: 2920-2927Abstract Full Text PDF PubMed Google Scholar) and to measure rates in NDP kinase (9Garces E. Cleland W.W. Kinetic studies of yeast nucleoside diphosphate kinase..Biochemistry. 1969; 8: 633-640Crossref PubMed Scopus (105) Google Scholar), we did not determine isotope effects.However, in 1975 Dexter Northrop discovered how to exploit the Swain-Schaad relationship between deuterium and tritium isotope effects (10Swain C.G. Stivers E.C. Reuwer Jr., J.F. Schaad L.J. Use of hydrogen isotope effects to identify the attacking nucleophile in the enolization of ketones catalyzed by acetic acid..J. Am. Chem. Soc. 1958; 80: 5885-5893Crossref Scopus (385) Google Scholar) to determine intrinsic isotope effects on the isotope-sensitive bond breaking step of an enzymatic reaction (11Northrop D.B. Steady-state analysis of kinetic isotope effects in enzymic reactions..Biochemistry. 1975; 14: 2644-2651Crossref PubMed Scopus (357) Google Scholar). The Swain-Schaad relationship says that the effect of tritium on a rate or equilibrium constant is the 1.442 power of the effect of deuterium substitution (this is derived from the relative masses of deuterium and tritium). Northrop assumed that there was no equilibrium isotope effect and thus that effects on V/K, the apparent first order rate constant at low substrate concentration and one of the independent kinetic constants, could be represented by Equation 1,D(V/K)=(V/K)H/(V/K)D=(Dk+c)/(1+c)(Eq. 1) where Dk = kH/kD, the intrinsic isotope effect on the bond breaking step, and c is a commitment to catalysis. If the equilibrium isotope effect is unity and there is only one isotope-sensitive step, Equation 1 is valid regardless of how many steps precede or follow the isotope-sensitive one.Northrop then subtracted one from each side of Equation 1 to get Equation 2.D(V/K)-1=(Dk-1)/(1+c)(Eq. 2) The equation for the tritium isotope effect is the same except that the superscripts are T rather than D. Then if one takes the ratios of Equation 2 for deuterium and tritium, one gets Equation 3.[D(V/K)-1]/[T(V/K)-1]=(Dk-1)/(Tk-1)(Eq. 3) However, because the Swain-Schaad relationship makes Tk = (Dk)1.442, one can substitute this value into Equation 3 to get an equation involving only experimental parameters and Dk.[D(V/K)-1]/[T(V/K)-1]=(Dk-1)/[(Dk)1.442-1](Eq. 4) Because this is a transcendental equation, one has to consult a table of values (12Cleland W.W. O'Leary M.H. Northrop D.B. Isotope Effects on Enzyme-catalyzed Reactions. University Park Press, Baltimore, MD1977: 280-283Google Scholar) or use a computer program to obtain a solution.At the time Dexter discovered these relationships, Mike Schimerlik in my laboratory was studying malic enzyme. We wanted to determine whether there was an equilibrium isotope effect on the reaction, so Mike proceeded to determine Keq values with unlabeled and 2-deuterated malate. He used the most accurate way to determine Keq, which is to make up reaction mixtures where the [products]/[reactants] ratio brackets Keq and then add enzyme. The ΔA that results as the reaction reaches equilibrium is plotted versus the [products]/[reactants] ratio, and the point where ΔA is zero is Keq. This worked well with unlabeled malate, but when Mike used deuterated malate, the A decreased greatly and then began to increase and returned to the starting point (Fig. 1). What he had forgotten was that he used unlabeled NADPH rather than deuterated nucleotide, which he didn't have. He thus discovered the equilibrium perturbation method for determining isotope effects on reversible reactions (13Schimerlik M.I. Rife J.E. Cleland W.W. Equilibrium perturbation by isotope substitution..Biochemistry. 1975; 4: 5347-5354Crossref Scopus (73) Google Scholar). The size of the perturbation is a function of the isotope effect although the relationship is only linear for small isotope effects.fractionalperturbation=[isotopeeffect-1]/2.72(Eq. 5) For isotope effects above 1.2, the complete equation must be used (14Cleland W.W. Measurement of isotope effects by the equilibrium perturbation technique..Methods Enzymol. 1980; 64: 104-125Crossref PubMed Scopus (111) Google Scholar). The fractional perturbation is the ratio of the perturbation size to the reciprocal of the sum of the concentrations of the perturbants (the molecules between which the label is exchanged). For malic enzyme, Mike was able to determine a deuterium isotope effect of 1.45 by equilibrium perturbation (1.47 on V/K by direct comparison) and also a13C isotope effect of 1.031, later confirmed by isotope ratio mass spectrometry (15Hermes J.D. Roeske C.A. O'Leary M.H. Cleland W.W. Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose-6-phosphate dehydrogenase..Biochemistry. 1982; 21: 5106-5114Crossref PubMed Scopus (206) Google Scholar).Mike determined that there was a sizable equilibrium isotope effect on the malic enzyme reaction (later refined to be 1.18 (16Cook P.F. Blanchard J.S. Cleland W.W. Primary and secondary deuterium isotope effects on equilibrium constants for enzyme-catalyzed reactions..Biochemistry. 1980; 21: 4853-4858Crossref Scopus (133) Google Scholar)) and thus that the equation for the isotope effect had to be expanded to allow for this.D(V/K)=(Dk+cf+crDKeq)/(1+cf+cr)(Eq. 6) The constants cf and cr are now commitments in forward and reverse directions. Each is the ratio of the rate constant for the bond breaking step to the net rate constant for release from the enzyme of the substrate whose V/K is involved or the first product released. In an equilibrium perturbation experiment, the commitments are for the release of the perturbants.The equation for the tritium isotope effect is the same except the leading superscripts are T rather than D. When one applies Northrop's method to these equations, the third term in the numerator does not cancel out, and thus one gets only an approximate answer. However, if one divides the experimental D(V/K) and T(V/K) isotope effects by the respective equilibrium isotope effects (which gives the values for the reverse reaction) and carries out the Northrop analysis one obtains an approximate value for Dk in the back reaction. Then the true Dk in the forward direction lies between the value determined in that direction and the one determined in the back reaction multiplied by the equilibrium isotope effect. This approach for malic enzyme gave limits of 5–8 for Dk in the forward reaction and 4–6.5 in the reverse direction (17Schimerlik M.I. Grimshaw C.E. Cleland W.W. Determination of the rate-limiting steps for malic enzyme by the use of isotope effects and other kinetic studies..Biochemistry. 1977; 16: 571-576Crossref PubMed Scopus (64) Google Scholar). The true value determined in 1985 by Chuck Grissom is 5.7 in the forward direction (18Grissom C.B. Cleland W.W. Use of intermediate partitioning to calculate intrinsic isotope effects for the reaction catalyzed by malic enzyme..Biochemistry. 1985; 24: 944-948Crossref PubMed Scopus (39) Google Scholar).When we first started to work on isotope effects none of us knew very much about them, but Jack Shiner at Indiana University steered us in the right direction, and we discovered that the physical organic chemists knew quite a bit about isotope effects. By attending the Gordon Conferences on isotopes (I have attended every one since 1981) we got to know all of the major players in the field and learned what they knew as well as returning the favor by giving them information about isotope effects on enzymes. This Gordon Conference meets every 2 years in California in the winter (alternating with the ad hoc Enzyme Mechanism Conference, which is convenient) and will meet next in Ventura on February 15–20, 2004. Anyone interested in isotope effects on enzymatic reactions should attend; students and postdoctoral fellows are welcome.As a result of our increasing interest in isotope effects, Marion O'Leary, Dexter Northrop, and I organized a Steenbock Symposium titled “Isotope Effects on Enzyme-catalyzed Reactions” here in Madison in 1976. This was very successful and the proceedings were published by University Park Press (19Cleland W.W. O'Leary M.H. Northrop D.B. Isotope Effects on Enzyme-catalyzed Reactions. University Park Press, Baltimore, MD1977: 280-283Google Scholar). This book includes computer programs for fitting isotope effect data and tables for use of Northrop's method and for equilibrium perturbation analysis.The year 1980 saw us publishing a number of measured equilibrium deuterium isotope effects (16Cook P.F. Blanchard J.S. Cleland W.W. Primary and secondary deuterium isotope effects on equilibrium constants for enzyme-catalyzed reactions..Biochemistry. 1980; 21: 4853-4858Crossref Scopus (133) Google Scholar) as well as kinetic isotope effects by John Blanchard on several enzymes (20Blanchard J.S. Cleland W.W. Use of isotope effects to deduce the chemical mechanism of fumarase..Biochemistry. 1980; 19: 4506-4513Crossref PubMed Scopus (86) Google Scholar, 21Blanchard J.S. Cleland W.W. Kinetic and chemical mechanisms of yeast formate dehydrogenase..Biochemistry. 1980; 19: 3543-3550Crossref PubMed Scopus (102) Google Scholar). The following year saw the development by Paul Cook in this laboratory of the theory for the variation of observed isotope effects with pH or the concentrations of other substrates (22Cook P.F. Cleland W.W. Mechanistic deductions from isotope effects in multireactant enzyme mechanisms..Biochemistry. 1981; 20: 1790-1796Crossref PubMed Scopus (162) Google Scholar, 23Cook P.F. Cleland W.W. pH variation of isotope effects in enzyme-catalyzed reactions. 1. Isotope- and pH-dependent steps the same..Biochemistry. 1981; 20: 1797-1805Crossref PubMed Scopus (125) Google Scholar, 24Cook P.F. Cleland W.W. pH variation of isotope effects in enzyme-catalyzed reactions. 2. Isotope-dependent step not pH dependent. Kinetic mechanism of alcohol dehydrogenase..Biochemistry. 1981; 20: 1805-1816Crossref PubMed Scopus (99) Google Scholar). The forward commitment in Equation 6 represents the ratio of the rate constant for the isotope-sensitive step to the net rate constant for release from the enzyme of the varied substrate in a direct comparison experiment, the labeled substrate in an internal competition experiment, or the perturbant in an equilibrium perturbation one. In an ordered mechanism where the forward commitment is for the first substrate, the observed isotope effect on V/Ka will be unity at infinite concentration of the second substrate, increasing to D(V/Kb) at very low levels of B. The value of D(V/Kb) on the other hand is independent of the level of A. In a random mechanism, saturation with one substrate does not eliminate the V/K isotope effect for the other one, although the value may change. This sort of experiment is very useful for determining the kinetic mechanism. Cook used these methods to show that NAD and cyclohexanol added in that order with liver alcohol dehydrogenase, whereas NAD and 2-propanol added randomly to the yeast enzyme (22Cook P.F. Cleland W.W. Mechanistic deductions from isotope effects in multireactant enzyme mechanisms..Biochemistry. 1981; 20: 1790-1796Crossref PubMed Scopus (162) Google Scholar).The effect of pH on observed isotope effects depends on whether the isotope-dependent and pH-dependent steps are the same. With a sticky substrate (one that reacts to give products faster than it dissociates), the isotope effect on V/K is reduced by an external forward commitment, but when one goes to a pH where the chemistry becomes rate-limiting, this external part of the forward commitment is eliminated (although any internal commitment remains), and so the V/K isotope effect increases (23Cook P.F. Cleland W.W. pH variation of isotope effects in enzyme-catalyzed reactions. 1. Isotope- and pH-dependent steps the same..Biochemistry. 1981; 20: 1797-1805Crossref PubMed Scopus (125) Google Scholar). With liver alcohol dehydrogenase, however, the V/K isotope effect for cyclohexanol was 2.5 at low and neutral pH but decreased above a pK of 9.4 to unity. In the reverse direction, the value for cyclohexanone was 2.1 at lower pH values and decreased above the same pK to 0.85 (24Cook P.F. Cleland W.W. pH variation of isotope effects in enzyme-catalyzed reactions. 2. Isotope-dependent step not pH dependent. Kinetic mechanism of alcohol dehydrogenase..Biochemistry. 1981; 20: 1805-1816Crossref PubMed Scopus (99) Google Scholar). In this mechanism, proton removal from the alcohol to give a zinc-bound alkoxide precedes hydride transfer, and above the pK this proton is lost to the medium, thus committing the reaction to continue. In the reverse direction at high pH hydride transfer comes to equilibrium waiting for a proton to be added from the solvent.In 1982 I was asked to write a review for Annual Reviews of Biochemistry on the use of isotope effects to elucidate enzyme mechanisms. I submitted the review, but it came back all marked up with many changes in wording and meaning, and after an unsatisfactory conversation with the redactor, I withdrew the paper. I then sent it to Critical Reviews in Biochemistry, where it was promptly accepted (25Cleland W.W. Use of isotope effects to elucidate enzyme mechanisms..CRC Crit. Rev. Biochem. 1982; 13: 385-428Crossref PubMed Scopus (232) Google Scholar).The next major advance in isotope effect theory was the use of multiple isotope effects by Jeff Hermes (15Hermes J.D. Roeske C.A. O'Leary M.H. Cleland W.W. Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose-6-phosphate dehydrogenase..Biochemistry. 1982; 21: 5106-5114Crossref PubMed Scopus (206) Google Scholar). When both isotope effects are on the same step, the effect of deuteration on13C isotope effects allows one to determine intrinsic isotope effects or narrow limits on them. Thus, in Equation 6 where the superscripts are 13, deuteration decreases the rate of the isotope-sensitive step and thus decreases the commitments by the size of the intrinsic deuterium isotope effect in the forward or reverse direction. If one uses both primary and secondary deuterium substitution, one has five equations (13C isotope effect with unlabeled, primary, and secondary deuterated substrates plus primary and secondary deuterium isotope effects) in five unknowns (three intrinsic isotope effects and the two commitments). Jeff applied this technique to glucose-6-P dehydrogenase both in water and in D2O (26Hermes J.D. Cleland W.W. Evidence from multiple isotope effect determinations for coupled hydrogen motion and tunneling in the reaction catalyzed by glucose-6-phosphate dehydrogenase..J. Am. Chem. Soc. 1984; 106: 7263-7264Crossref Scopus (51) Google Scholar). The intrinsic13C isotope effect was 4% in both solvents, but the intrinsic primary deuterium isotope effect was 5.3 in water and 3.7 in D2O. The α-secondary deuterium isotope effect also decreased in D2O. However, the surprise was that the sum of forward and reverse commitments increased from 1.24 in water to 2.5 in D2O with most of the change being in the forward commitment. Thus the major effect of D2O was on the conformation changes that precede the chemical step rather than on the chemistry itself. However, the decreased intrinsic deuterium isotope effects in D2O reflect the coupled hydrogen motions in the transition state (proton from the 1-hydroxyl going to aspartate on the enzyme, hydride going from C-1 of glucose-6-P to C-4 of NADP, hydrogen at C-4 of NADP going from trigonal to tetrahedral; Fig. 2). This coupled motion effect, where the first deuterium substitution decreases the effect of further deuteration, shows that tunneling is involved in the hydrogen motions.Fig. 2Transition state for the glucose-6-P dehydrogenase reaction with arrows showing coupled hydrogen motions (26Hermes J.D. Cleland W.W. Evidence from multiple isotope effect determinations for coupled hydrogen motion and tunneling in the reaction catalyzed by glucose-6-phosphate dehydrogenase..J. Am. Chem. Soc. 1984; 106: 7263-7264Crossref Scopus (51) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)This coupled motion effect is very prominent in the formate dehydrogenase reaction (27Hermes J.D. Morrical S.W. O'Leary M.H. Cleland W.W. Variation of transition-state structure as a function of the nucleotide in reactions catalyzed by dehydrogenases. 2. Formate dehydrogenase..Biochemistry. 1984; 23: 5479-5488Crossref PubMed Scopus (133) Google Scholar). The deuterium isotope effect at C-4 of the ring of NAD was 1.23 despite the fact that the equilibrium isotope effect is 0.89. This shows that the motion of this hydrogen from trigonal to tetrahedral is coupled into the reaction coordinate so that there is little or no restoring force at the transition state. However, if deuterated formate is used, this secondary isotope effect decreases to 1.07, showing that the first deuterium substitution decreases the effect of the second deuteration. This is a nice system to study because there are no commitments (the13C isotope effect in formate is independent of deuteration). When the nucleotide substrate was changed from NAD to thio-NAD and then to acetylpyridine-NAD, the transition state became earlier as the redox potential of the nucleotide became more positive. This led to larger primary deuterium isotope effects (2.17 to 2.60 to 3.32), smaller secondary deuterium isotope effects (1.23 to 1.18 to 1.06) as the coupling of the secondary motion into the reaction coordinate decreased, and smaller13C isotope effects (4.2 to 3.8 to 3.6%) as the degree of C–C cleavage decreased. The secondary isotope effects decreased halfway to the equilibrium isotope effect with deuterated formate (1.07 to 1.03 to 0.95). Thus with multiple isotope effects one can really determine transition state structure.When a deuterium and the13C isotope effect are on different steps, deuteration makes the deuterium-sensitive step more rate-limiting and thus increases one of the commitments for the 13C-sensitive step so that the observed13C isotope effect decreases. Further, the three measured isotope effects are not independent. In the direction where the deuterium-sensitive step comes first, the equation is,[13(V/K)H-1]/[13(V/K)D-1]=D(V/K)/DKeq(Eq. 7) although in the reverse direction where the13C-sensitive step comes first,[13(V/K)H-13Keq]/[13(V/K)D-13Keq]=D(V/K)(Eq. 8) These equations are really the same except that the first is expressed in terms of the parameters for the forward reaction, and the second one includes the parameters for the reverse reaction. When these equations were applied to data for malic enzyme, the data fitted Equation 7, but not Equation 8, showing that dehydrogenation precedes decarboxylation (15Hermes J.D. Roeske C.A. O'Leary M.H. Cleland W.W. Use of multiple isotope effects to determine enzyme mechanisms and intrinsic isotope effects. Malic enzyme and glucose-6-phosphate dehydrogenase..Biochemistry. 1982; 21: 5106-5114Crossref PubMed Scopus (206) Google Scholar).6-Phosphogluconate dehydrogenase was also shown to catalyze a stepwise reaction (28Rendina A.R. Hermes J.D. Cleland W.W. Use of multiple isotope effects to study the mechanism of 6-phosphogluconate dehydrogenase..Biochemistry. 1984; 23: 6257-6262Crossref PubMed Scopus (67) Google Scholar), but prephenate dehydrogenase provided some surprises (29Hermes J.D. Tipton P.A. Fisher M.A. O'Leary M.H. Morrison J.F. Cleland W.W. Mechanisms of enzymatic and acid-catalyzed decarboxylations of prephenate..Biochemistry. 1984; 23: 6263-6275Crossref PubMed Scopus (62) Google Scholar). The isotope effects were measured with a substrate lacking the keto group in the side chain but having a Vmax of 78% and V/K of 18% that of prephenate. The13C isotope effect in the CO2 product was 1.03% with deuterated substrate but only 0.33% with unlabeled substrate, whereas the deuterium isotope effect on hydride transfer was 2.34. Thus the reaction is concerted with intrinsic13C and deuterium isotope effects of 1.0155 and 7.3 and a forward commitment of 3.7, assuming no reverse commitment for the irreversible reaction. The deuterium isotope effect is large, showing considerable C–H cleavage in the transition state, but the 1.55%13C isotope effect shows that the reaction is asynchronous with little C–C cleavage in the transition state. The reason the reaction is concerted is presumably that the energy of aromatization is so great that the putative keto intermediate has no stability. In fact, if one removes one double bond from the ring of the prephenate analog with no ketone in the side chain the product of the reaction is a ketone and no decarboxylation takes place. The enzyme is thus a secondary alcohol dehydrogenase, and the decarboxylation takes place because of the instability of the keto product.The multiple isotope method was also applied to15N and deuterium isotope effects in studies on phenylalanine ammonia lyase (30Hermes J.D. Weiss P.M. Cleland W.W. Use of nitrogen-15 and deuterium isotope effects to determine the chemical mechanism of phenylalanine anmmonia-lyase..Biochemistry. 1985; 24: 2959-2967Crossref PubMed Scopus (152) Google Scholar), adenosine deaminase (31Weiss P.M. Cook P.F. Hermes J.D. Cleland W.W. Evidence from nitrogen-15 and solvent deuterium isotope effects on the chemical mechanism of adenosine deaminase..Biochemistry. 1987; 26: 7378-7384Crossref PubMed Scopus (72) Google Scholar), and aspartate aminotransferase (32Rishavy M.A. Cleland W.W. 13C and15N kinetic isotope effects on the reaction of aspartate aminotransferase and the tyrosine-225 to phenylalanine mutant.Biochemistry. 2000; 39: 7546-7551Crossref PubMed Scopus (17) Google Scholar) to provide details of the mechanisms of these enzymes. The next development of the theory came with the discovery of intermediate partitioning by Chuck Grissom (18Grissom C.B. Cleland W.W. Use of intermediate partitioning to calculate intrinsic isotope effects for the reaction catalyzed by malic enzyme..Biochemistry. 1985; 24: 944-948Crossref PubMed Scopus (39) Google Scholar). With malic enzyme one can add oxaloacetate and NADPH and regenerate the putative intermediate on the enzyme. This will then partition both back to malate and NADP and forward to CO2 and pyruvate. The partitioning ratio (pyruvate/malate = 0.47) is the forward commitment for the decarboxylation step and allowed determination of the intrinsic13C isotope effect as 1.044. With the deuterium and tritium V/K isotope effects, the equations allow one to determine the intrinsic deuterium isotope effect as 5.7, the forward commitment to hydride transfer as 3.3, and the ratio of reverse hydride transfer to decarboxylation as 10 (18Grissom C.B. Cleland W.W. Use of intermediate partitioning to calculate intrinsic isotope effects for the reaction catalyzed by malic enzyme..Biochemistry. 1985; 24: 944-948Crossref PubMed Scopus (39) Google Scholar). Thus the tools are available to dissect the entire mechanism.As the decade of the 90s dawned, we began to determine18O and other isotope effects to study phosphoryl and acyl transfer. It is very difficult to extract the oxygen out of phosphate quantitatively and insert it into CO2, so we adopted the use of the remote label method, which had been pioneered by Marion O'Leary (33O'Leary M.H. Marlier J.F. Heavy-atom isotope effects on the alkaline hydrolysis and hydrazinolysis of methyl benzoate..J. Am. Chem. Soc. 1979; 101: 3300-3306Crossref Scopus (131) Google Scholar). For example, to measure the secondary18O isotope effect on the hydrolysis of glucose-6-P, one prepares two versions of this molecule. One has13C at C-1 and three18O's in the phosphate group. The second has12C at C-1 and no other labels. By12C we mean carbon that has the 1% natural abundance of13C removed (it is the by-product of making13C). One then mixes 1% of the former with 99% of the latter to get a solution of glucose-6-P with the natural abundance of13C at C-1 but with every13C accompanied by three18O's. This remote labeled material is then used in a reaction, and the residual glucose-6-P and the glucose product (phosphorylated back to glucose-6-P by hexokinase) are then degraded by glucose-6-P and 6-P-gluconate dehydrogenases to CO2 and ribulose-5-P. Analysis of the CO2 reveals the isotopic discrimination between the two species in the substrate mixture. One then uses glucose-6-P with no special labels in the same experiment, and this determines any13C isotope effect at C-1. Division of the apparent isotope effect for the remote labeled substrate by the value from natural abundance substrate gives the desired18O isotope effect.The remote label method is very powerful and allows one to determine almost any isotope effect in any position of a molecule as long as there is a carbon that can be isolated as CO2 or a nitrogen that can be isolated and converted to N2. If there is only one nitrogen in a molecule this is simple as samples can be sealed in quartz tubes with CuO and heated to convert all organic matter to CO2, H2O, and N2, which are readily separated for analysis of N2 by the isotope ratio mass spectrometer. Convenient remote labels are nitro groups of p-nitrophenol or m-nitrobenzyl alcohol (inserted by nitration of triphenyl phosphate followed by hydrolysis or benzaldehyde followed by reduction, using either 15N- or 14N-labeled nitrate). Another useful remote label is the exocyclic amino group of adenine, which is readily inserted by the reaction of ammonia with chloropurine riboside and removed later for analysis by adenosine deaminase. This allows ATP, NAD, or other adenine-containing molecules to be remote-labeled.Al Hengge and others in the laboratory carried out extensive measurements of18O isotope effects on phosphoryl transfer using the remote label method for analysis. This showed that phosphate monoesters have dissociative transition states for their reactions, diesters have Sn2 reactions, and triesters have associative transition states although they do not form phosphorane intermediates unless geometry requires this (34Cleland W.W. Hengge A.C. Mechanisms of phosphoryl and acyl transfer..FASEB J. 1995; 9: 1585-1594Crossref PubMed Scopus (110) Google Scholar).Al Hengge and Rob Hess carried out a thorough study of the reactions of p-nitrophenyl acetate with various nucleophiles (35Hengge A.C. Hess R.A. Concerted or stepwise mechanisms for acyl transfer reactions of p-nitrophenyl acetate? Transition state structures from isotope effects..J. Am. Chem. Soc. 1994; 116: 11256-11263Crossref Scopus (121) Google Scholar) and with several enzymes (36Hess R.A. Hengge A.C. Cleland W.W. Isotope effects on enzyme-catalyzed acyl transfer from p-nitrophenyl acetate: concerted mechanisms and increased hyperconjugation in the transition state..J. Am. Chem. Soc. 1998; 120: 2703-2709Crossref Scopus (51) Google Scholar). In opposition to what most textbooks say, these reactions do not have a tetrahedral intermediate but are concerted. Only when the leaving group has a pK of 16 or higher does a tetrahedral intermediate form and 18O exchange take place during hydrolysis. Al and Rob used five isotope effects to study these reactions. The nitro group was the remote label for measurement of the other isotope effects, and the15N isotope effect i" @default.
• W2031569498 created "2016-06-24" @default.
• W2031569498 creator A5033718331 @default.
• W2031569498 date "2003-12-01" @default.
• W2031569498 modified "2023-10-18" @default.
• W2031569498 title "The Use of Isotope Effects to Determine Enzyme Mechanisms" @default.
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• W2031569498 doi "https://doi.org/10.1074/jbc.x300005200" @default.
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