Progress 01/01/01 to 09/30/11
Outputs
OUTPUTS: Sir2 The synthesis of 12C, 16O, and 13C, 18O labeled Na propionates has been completed. The two compounds were mixed together to natural abundance levels (determined by IRMS) and the compound has been incorporated into an 11-mer peptide. This will be used for the determination of the KIE of the nucleophilic O in the Sir2 reaction. A small amount of 13C, 18O labeled Na propionate (5-10 nanomoles) was incorporated into the 11-mer for MALDI-TOF analysis to confirm that the process of incorporation does not cause the loss of 18O in the propionate. The analysis showed that the amount of 18O labeling for the propionate portion of the 11-mer is consistent with that of the original propionate (98% singly labeled 18O, 75% doubly labeled 18O) indicating there is no loss of labeling during the peptide synthesis. The KIE studies for the nucleophilic attack of O are now underway. NAD synthetase (M. tuberculosis) is a multifunctional enzyme containing both a glutaminase and a synthetase domain. In order to study the chemical steps after the initial glutamine hydrolysis step, the isotope effects are first being determined using natural abundance ammonia. Residual ammonia from the partial reactions was removed by steam distillation and oxidized to N2(g) with hypobromite. The 15N/14N ratio in the N2(g) was analyzed on an isotope ratio mass spectrometer after collection on high vacuum lines. The N-15 isotope effect on ammonia in H2O is 1.34 +- 0.08 %. This isotope effect indicates the amidation step is rate limiting. Specifically, the formation of the tetrahedral intermediate is rate limiting rather than its breakdown. The N-15 isotope in D2O is 2.0 +- 0.1 % indicating a proton is being concomitantly transferred from the attacking ammonia to form the amide in this step. PARTICIPANTS: Mark Anderson, Associate Scientist; Laurie Reinhardt, Associate Scientist TARGET AUDIENCES: The target audience for this work is mechanistic enzymologists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These studies contribute to our understanding of how the enzymes studied actually catalyze their reactions.
Publications
- 13C Isotope Effect on the Reaction Catalyzed by Prephenate Dehydratase. J. Van Vleet, A. Kleeb, P. Kast, D. Hilvert and W. W. Cleland. Biochem. Biophys. Acta, 1804, 752-754 (2010)
- The low-barrier hydrogen bond in enzymic catalysis. W. W. Cleland. Adv. Phys. Org. Chem. 44, 1-17 (2010).
- A Kinetic and Isotope Effect Investigation of the Urease-Catalyzed Hydrolysis of Hydroxyurea. J. F. Marlier, L. I. Robins, K.A. Tucker, J. Rawlings, M.A. Anderson and W. W. Cleland. Biochemistry, 49, 8213-8210 (2010).
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: 1) We have determined the isotope effects in N-1 of nicotinamide during the initial step of catalysis by Sir2 using the acetyl (1.0122 +-0.0004), hydroxyacetyl (1.0281 +-1.0011), and propionyl (1.0305 +-0.0016) peptides. The propionyl analog has the highest KIE which we feel is very close to the intrinsic effect. The C1 carbon effect will be determined for each analog by oxidation of the residual ribose with bromine/periodate to produce CO2 from C-1. 2) Previously we reported the C and N KIE for nicotinamide and other substrates for nicotinamidase as well as several mutants. The final C KIE values are being determined for the substrates 5-methylnicotinamide and thionicotinamide. A crystal structure of this enzyme from E.Coli has also been determined and will be included in the future publication. 3) Preliminary experimental procedures have been performed in preparation for the measurement of 15N isotope effects on glutamine hydrolysis by NAD Synthetase from M. tuberculosis. Measuring the isotope effects includes performing the enzymatic reaction, removing enzyme, purifying residual glutamine, hydrolyzing the residual glutamine, oxidizing the resulting ammonia, and measuring the 15N/14N ratio in the ammonia by isotope ratio mass spectroscopy. The 15N/14N ratio of the unreacted glutamine substrate is also necessary to calculate Ro needed for determining the isotope effects. The Ro of glutamine substrate to be used in the enzymatic reactions was -7.8 +- 0.3. PARTICIPANTS: Mark Anderson, Associate Scientist; Laurie Reinhardt, Associate Scientist TARGET AUDIENCES: The results from this project are published in the biochemical literature for the benefit of interested scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
These results help our understanding of how enzymes work.
Publications
- Symbiotic Nitrogen Fixation in the Fungus Gardens of Leaf-Cutter Ants. A. A. Pinto-Tomas, M. A. Anderson, G. Suen, D. M. Stevenson, F. S. T. Chu, W. W. Cleland, P. J. Weimer and C. R. Currie. Science, 326, 1120-1123 (2009).
- 13C Isotope Effect on the Reaction Catalyzed by Prephenate Dehydratase. J. Van Vleet, A. Kleeb, P. Kast, D. Hilvert and W. W. Cleland. Biochem. Biophys. Acta, in press (2009).
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The kinetic parameters and KIEs for the reaction catalyzed by nicotinamidase using different substrates have been studied. The enzymatic reactions were run to known fraction of reaction values and were then acid quenched. The enzyme was filtered away and the products of the reaction, ammonia, substrate nicotinate, and residual substrate, were separated on an AG1-X8 column. The residual substrate was converted to nicotinate by base hydrolysis and the ammonia was trapped in a dilute sulfuric acid solution. The product ammonia and ammonia from the residual substrate were further purified by steam distillation. Each was then oxidized with NaOBr and the resultant nitrogen gas was analyzed by IRMS to give the Rp and Rs values needed to obtain the KIE. The primary N-15 isotope effects ranged from 1.0121 with nicotinamide to 1.0231 with pyrazinamide, while kcat and kcat/Km values are not very different. The reaction has also been studied using the D51A and D51N mutants of nicotinamidase with nicotinamide as the substrate. The aspartic acid mutated is believed to chelate the Zn center. The reaction catalyzed by the D51A mutant has a KIE of 1.0166 indicating that the forward commitment is decreased in this system also. The D51A mutant likely decreases the binding of the substrate. The reaction catalyzed by the D51N mutant has a KIE of 1.0045 indicating that the forward commitment has increased possibly due to the substrate being bound more tightly. PARTICIPANTS: Participants are: John Marlier (visiting professor), Jill Rawlings (Visiting Professor), Laurie Reinhardt (Associate Scientist), Mark Anderson (Associate Scientist) and Jeremy Van Vleet (Grad Student). TARGET AUDIENCES: Target audience would be mechanistic enzymologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
This project continues to use kinetic studies to determine enzyme mechanisms and contribute to our knowledge of how these masterful catalysts accelerate reactions.
Publications
- Use of Isotope Effects to Determine Enzyme Mechanisms. W. W. Cleland, J Label Compd Radiopharm 50, 1006-1015 (2007).
- Carbon Isotope Effect Study on Orotidine 5'-Monophosphate Decarboxylase Support for an Anionic Intermediate. J. Van Vleet, L. Reinhardt, B. Miller, A. Sievers, W. Cleland, Biochemistry 47, 798-803 (2008).
- A Heavy-Atom Isotope Effect and Kinetic Investigation of the Hydrolysis of Semicarbazide by Urease from Jack Bean (Canavalia ensiformis). J. F. Marlier, E. J. Fogle and W. W. Cleland. Biochemistry 47, 11158-11163 (2008)
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: In studies of the urease catalyzed hydrolysis of semicarbazide, the N-15 isotope effect in the NH2 end was 1.0009, while in the hydrazine end it was 1.0045. This suggests that the initial leaving group is hydrazine, probably protonated. Further, the 1.0045 value may be a mixture of a primary effect that is several percent normal for the nitrogen next to the carbonyl group and an inverse effect due to protonation at the other nitrogen. The C-13 isotope effect on the reaction is 1.0357, which suggests that the chemistry is fully rate limiting. We have also determined isotope effects on urease-catalyzed hydrolysis of hydroxyurea. The reaction is biphasic with a burst followed by a slower steady state rate. We believe this results from the enzyme-substrate complex reversibly, but slowly, forming a non-productive complex with the NHOH group bound to Ni. Preliminary isotope effects are 1.0014 in the NH2 group, 1.0011-1.0023 in the NHOH group and 1.0134-1.0189 in the carbonyl
carbon, with the lower values in the burst and the higher ones in the steady state. Clearly there are commitments here, and the chemistry is not fully rate limiting
PARTICIPANTS: Participants are: John Marlier (visiting professor), Jill Rawlings (Visiting Professor), Laurie Reinhardt (Associate Scientist), Mark Anderson (Assistant Scientist) and Jeremy Van Vleet (Grad Student).
TARGET AUDIENCES: Target audience would be mechanistic enzymologists
Impacts
This project continues to use kinetic studies to determine enzyme mechanisms and contribute to our knowledge of how these masterful catalysts accelerate reactions.
Publications
- Investigating the Roles of Putative Active Site Residues in the Oxalate Decarboxylase from Bacillus subtilis. D. Svedruzic, Y. Liu, L. A. Reinhardt, E. Wroclawska W. W. Cleland, and N. G. J. Richards. Arch. Biochem. Biophys. 464, 36-47 (2007).
- Insights into the Mechanism of Flavoprotein-Catalyzed Amino Oxidation from Nitrogen Isotope Effects on the Reaction of N-Methyltrptophan Oxidase. E.C. Ralph, J. S. Hirschi, M. A. Anderson, W. W. Cleland, D. A. Singleton and P. F. Fitzpatrick. Biochemistry, 46, 7655-7664 (2007).
- Carbon Isotope Effect Study on Orotidine 5'-Monophosphate Decarboxylase: Support for an Anionic Intermediate. J. Van Vleet, L. Reinhardt, B. Miller, A. Sievers, W. Cleland, Biochemistry, in press (2007).
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Progress 01/01/06 to 12/31/06
Outputs
The N-15 isotope effect for the conversion of ethanolamine to acetaldehyde and ammonia by ethanolamine ammonia lyase has been determined with deuterated ethanolamines. With 1,1-deuterated ethanolamine (gives a primary deuterium isotope effect) the value was 1.0007 +- 0.0002, compared with 1.0013 +- 0.0004 with unlabeled ethanolamine. This is consistent with C-N cleavage being a different step from initial C-H cleavage and deuterium substitution making C-N cleavage less rate limiting. With 2,2-deuterated ethanolamine the N-15 isotope effect was 1.0038 +- 0.0003. This points to a very large secondary deuterium isotope effect approaching 3.0, which in turn suggests that C-N cleavage produces a cation radical with a very low fractionation factor for the hydrogens at C-2. When all positions in ethanolamine are deuterated, the N-15 isotope effect is 1.0016 +- 0.0003, which shows that the effects of deuterium substitution at C-1 and at C-2 are canceling each other. The N-15
isotope effect with alanine as a substrate for tryptophan 2-monooxygenase was 1.0145 +- 0.0007, or when corrected for deprotonation of the alanine, 0.9917. Since the deuterium isotope effect at the alpha position is 6 and is pH independent, the N-15 value is consistent with the oxidation of alanine occurring through a hydride transfer mechanism. Primary C-13 isotope effects have been determined for several substrates of OMP decarboxylase and its K59A mutant. -------------------------------------------------------------- Table 1: Primary C-13 Kinetic Isotope Effects for the exocyclic -CO2 of Orotidine-5'-monophosphate Decarboxylase in 100 mM MES, 5 mM DTT, pH 6.5 Enzyme Substrate C-13 IE +- SE wt OMP 1.0255 +- 0.0004 K59A OMP 1.0541 +- 0.0004 wt FOMP 1.0105 +- 0.0002 K59A FOMP 1.0356 +- 0.0001 wt 2dOMP 1.0461 +- 0.0006 The K59A mutant has a V/K value 5 orders of magnitude slower than the WT enzyme and the large values for the isotope effects show that the chemistry is rate limiting.
With the 5-fluoro-OMP substrate which has the same V/K value as OMP with WT enzyme, the transition state appears to be earlier than with OMP, as one would expect from the inductive effect of the fluorine. 2-deoxy-OMP gives a V/K value 2000-fold less than OMP and again the chemistry appears to be completely rate limiting. In studies of the urease catalyzed hydrolysis of semicarbazide, the N-15 isotope effect in the NH2 end was 1.0009, while in the hydrazine end it was 1.0045. This suggests that the initial leaving group is hydrazine, possibly protonated. Further, the 1.0045 value may be a mixture of a primary effect that is several percent normal for the nitrogen next to the carbonyl group and an inverse effect due to protonation at the other nitrogen. We still have to measure the C-13 isotope effect on the reaction.
Impacts
Work from this project has led to the development of many kinetic methods for study of enzymatic reactions. Our work with heavy atom isotope effects allows us to determine transition state structures for non-enzymatic as well as enzymatic reactions. Since transition state analogs are often used as medically important drugs, it is important to know what the transition states for enzymatic reactions look like.
Publications
- 13C and 15N Isotope Effects for the Conversion of L-Dihydroorotate to N-Carbamyl-L-aspartate Using Dihydroorotase from Hamster and Bacillus caldolyticus. M. A. Anderson, W. W. Cleland, D. T. Huang, C. Chan, M. Shojaei, and R. I. Christopherson. Biochemistry, 45, 7132-7139 (2006).
- Probing Nitrogen Sensitive Steps in the Free Radical-Mediated Deamination of Amino Alcohols by Ethanolamine Ammonia-Lyase. R. R. Poyner, M. A. Anderson, V. Bandarian, W. W. Cleland and G. H. Reed. J. Am. Chem. Soc. 128, 7120-7121 (2006).
- A Multiple Isotope Effect Study of the Hydrolysis of Formamide by Urease from Jack Bean (Canavalia ensiformis). J. F. Marlier and W. W. Cleland. Biochemistry, 45, 9940-9948 (2006).
- Mechanistic Studies of the Flavoenzyme Tryptophan 2-Monooxygenase: Deuterium and 15N Kinetic Isotope Effects on Alanine Oxidation by an L-Amino Acid Oxidase. E. C. Ralph, M. A. Anderson, W. W. Cleland and P. F. Fitzpatrick. J. Am. Chem. Soc. 128, in press (2006).
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Progress 01/01/05 to 12/31/05
Outputs
1) Isotope effects have been measured on the hydrolysis of formamide catalyzed by urease. The carbonyl carbon 13C isotope effect of 1.0241 and the 15N isotope effect in the ammonia product show that the chemistry is rate limiting and that breakdown of the tetrahedral intermediate is the slow step. The formyl hydrogen deuterium isotope effect of 0.95 and the 18O one in the carbonyl oxygen of 0.9980 are consistent with equilibrium isotope effects for formation of the tetrahedral intermediate. The 18O isotope effect in the attacking nucleophile is 2.22% inverse, again consistent with an equilibrium isotope effect for forming the tetrahedral intermediate. Formamide hydrolysis has a lower pH optimum than that for urea. It appears that the carbonyl oxygen is bound to Ni1, while OH is bound to Ni2. His320 must be protonated in order to protonate the NH2 group becoming ammonia, so there is reverse protonation between it and the Ni-bound OH. Cys319 is peferentially ionized for
reaction of urea (H-bonding to the second NH2?), but protonated for reaction of formamide, explaining the pH profiles. 2) Isotope effects have been determined for the conversion of dihydroorotate to carbamoylaspartate catalyzed by dihydroorotase. The primary 13C and 15N isotope effects on the C-N cleavage were 1%, while the secondary 13C isotope effect in the carbamoyl group was 0.2% . Thus the chemistry is at least partly rate limiting. The equilibrium isotope effects (0.5% primary 15N, 0.2% primary 13C, 0.2% secondary 13C) are consistent with stiffer bonding in the ring than in the open chain product. 3) The 15N isotope effect for the cleavage of propanolamine catalyzed by ethanolamine ammonia lyase was 0.12% for the 2R isomer and 0.55% for the 2S one, compared with 0.13% for ethanolamine. The product in both cases is a 2S isomer, so rotation around the C1-C2 bond is required for the 2R isomer and not the 2S one. This rotation (which also occurs with ethanolamine) is partly rate
limiting and thus reduces the observed isotope effects for the 2R isomer and for ethanolamine. 4) Kinetic studies were carried out on L. lactis galactokinase for wild type and His-tagged enzyme and D183N, D183A, R35K and R36A mutants. The Asp183 mutants are inactive, suggesting that it is the general base in the reaction. The R36 mutants have activity reduced by 25 (K) or 75 (A). These groups may help stabilize the transition state.
Impacts
Work from this project has led to the development of many kinetic methods for study of enzymatic reactions. Our work with heavy atom isotope effects allows us to determine transition state structures for non-enzymatic as well as enzymatic reactions. Since transition state analogs are often used as medically important drugs, it is important to know what the transition states for enzymatic reactions look like.
Publications
- Multiple Isotope Effect Study of the Acid-Catalyzed Hydrolysis of Methyl Formate. J. F. Marlier, T. G. Frey, J. A. Mallory & W. W. Cleland. J. Org. Chem. 70, 1737 (2005).
- Kinetic Analysis of the L-Ornithine Transcarbamoylase from Pseudomonas savastanoi pv. phaseolicola that is Resistant to the Transition State Analogue (R)-N?-(N'-sulfodiaminophosphinyl)-L-ornithine. M. D. Templeton, L. A. Reinhardt, C. A. Collyer, R. E. Mitchell & W. W. Cleland. Biochemistry 44, 4408 (2005).
- Kinetic and structural analysis of ?-D-glucose-1-phosphate cytidylyltransferas from Salmonella typhi. N. M. Koropatkin, W.W. Cleland and H. M. Holden. J. Biol. Chem. 280, 10774-10780 (2005).
- Isotope ffects on the Enzymatic and Non-Enzymatic Reactions of Chorismate. S. K. Wright, M. S. DeClue, A. Mandal, L. Lee, O. Wiest, W. W. Cleland and D. Hilvert. J. Am. Chem. Soc. 217, 12957-12964 (2005).
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Progress 01/01/04 to 12/31/04
Outputs
1. We have measured further C-13 and O-18 isotope effects on the reaction catalyzed by oxalate decarboxylase. 2. We have prepared an E. coli strain that lacks both asparagine synthetases so that we can use it to prepare specific mutants of this enzyme without a background of wild type enzymes. 3. We have measured N-15 and C-13 isotope effects in reactions catalyzed by dihydroorotase and arginine kinase.
Impacts
Work from this project has led to the development of many kinetic methods for study of enzymatic reactions. Our work with heavy atom isotope effects allows us to determine transition state structures for non-enzymatic as well as enzymatic reactions. Since transition state analogs are often used as medically important drugs, it is important to know what the transition states for enzymatic reactions look like.
Publications
- High resolution X-ray structure of dTDP-glucose 4,6-dehydratase from Streptomyces venezuelae. S.T. Allard, W. W. Cleland & H.M. Holden. J. Biol. Chem. 279, 2211-2220 (2004).
- Substrate Binding, Catalysis, and Product Release. W. W. Cleland. in Encyclopedia of Biological Chemistry (W. J. Lennarz & M. D. Lane, eds), Elsevier, Oxford, Vol 4, pp. 123-126 (2004).
- The use of isotope effects to determine enzyme mechanisms. W. W. Cleland. Arch. Biochem. Biophys. 433, 2-12 (2005).
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Progress 01/01/03 to 12/31/03
Outputs
We have begun a study of asparagine synthetase B, using the C1S mutant which no longer uses glutamine as a substrate, but does use ammonia. The N-15 isotope effect in ammonia, corrected for the state of protonation of ammonia is 0.985 +/- 0.001, compared with an equilibrium isotope effect of 0.995. Thus the nitrogen is more stiffly bonded than in either ammonia or asparagine during the limiting step. The kinetic C-13 isotope effect at the beta-carbon of aspartate is 1.0014, while the equilibrium isotope effect is 1.0005. It appears that the reaction of MgATP and aspartate may commit the latter to undergo the reaction. Or, since 100 mM ammonium chloride was present (pH 8), possibly the high ammonia level committed the reaction. We have completed our measurements of isotope effects on the enzymatic and non-enzymatic reactions of chorismate in its mutase reaction. In the non-enzymatic reaction we discovered that the reaction goes twice as fast in hydroxylamine buffer at
pH6 than in other buffers, although the product distribution is similar. The reason for this is not clear. For comparison with our studies on oxalate decarboxylase, we have begun a similar study of oxalate oxidase, which converts oxalate and oxygen into carbon dioxide and hydrogen peroxide. Conditions for measuring the O-18 and C-13 isotope effects have been worked out and numbers should be available shortly. With a K59A mutant of OMP decarboxylase with a V/K value 5 orders of magnitude reduces, the C-13 isotope effect is 1.054. With wt enzyme and 5-F-OMP, the value is only 1.010. This is expected because the inductive effect of the flourine makes the transition state earlier and these data support the mechanism involving a short lived carbanion intermediate. With 2'-deoxy-OMP, the value is 1.054, so the chemistry is fully rate limiting.
Impacts
Work from this project has led to the development of many kinetic methods for study of enzymatic reactions. Our work with heavy atom isotope effects allows us to determine transition state structures for non-enzymatic as well as enzymatic reactions. Since transition state analogs are often used as medically important drugs, it is important to know what the transition states for enzymatic reactions look like.
Publications
- Metal Ion Catalyzed Hydrolysis of Ethyl p-Nitrophenyl Phosphate. J. Rawlings, W. W. Cleland and A. C. Hengge. J. Inorg. Biochem. 93, 61-65 (2003).
- Heavy Atom Isotope Effects on the Reaction Catalyzed by Oxalate Decarboxylase from Bacillus subtilis. L. A. Reinhardt, D. Svedruzic, C. H. Chang, W. W. Cleland and N. G. R. Richards. J. Am. Chem. Soc. 125, 1244-1252 (2003).
- 2H, 13C, and 15N Kinetic Isotope Effects on the Reaction of the Ammonia-Rescued K258A Mutant of Aspartate Aminotransferase. S. K. Wright, M. A. Rishavy and W. W. Cleland. Biochemistry 42, 8377-8376 (2003).
- Reverse Protonation is the Key to General Acid-Base Catalysis in Enolase. P. A. Sims, T. M. Larsen, R. R. Poyner, W. W. Cleland and G. H. Reed. Biochemistry 42, 8298-8306 (2003).
- The Use of Isotope Effects to Determine Enzyme Mechanisms. W. W. Cleland. J. Biol. Chem. 278, 51975-51984 (2003).
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Progress 01/01/02 to 12/31/02
Outputs
C-13 and O-18 isotope effects on oxalate decarboxylase show that the first step is removal of a proton and an electron to give an oxalate radical which then decarboxylates to give a formate radical anion that becomes formate. Similar isotope effects on chorismate mutase show that the non-enzymatic reaction has a concerted reaction similar to that of the enzymatic one. The N-15 isotope effect in ammonia with asparagine synthetase B is 1.5% inverse, suggesting the bonding to the nitrogen is increasing in the rate limiting transition state.
Impacts
These studies show the power of isotope effect studies to determine the mechanisms of enzymatic reactions.
Publications
- Snider, M. J., Reinhardt, L., Wolfenden, R. and Cleland, W. W. 2002. N-15 Kinetic Isotope Effects on Uncatalyzed and Enzymatic Deamination of Cytidine. Biochemistry 41:415-421.
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Progress 01/01/01 to 12/31/01
Outputs
Mechanism of phosphoryl transfer: The primary O-18 isotope for reaction of glucose and MgATP in the presence of yeast hexokinase is 1.0013 at pH 5.3, suggesting that the chemistry is not fully rate limiting, even at this low pH. We will use 1/5-anhydroglucitol (a slow substrate) to try to make the chemistry more rate limiting. The compound 2-[4-nitrophenoxy)]ethyl triphosphate has been shown to be an alternate substrate for myosin ATPase. We will use it to determine primary O-18 isotope effects in an attempt to find conditions where the chemistry is rate limiting. Primary and secondary O-18 isotope effects were determined for the alkaline hydrolysis of diethyl phosphotriesters with choline or m-nitrobenzyl alcohol as leaving groups. The primary isotope effects were 4-5%, and the secondary 3.3%. These values were interprested as resulting from a highly associative transition state with a large reaction coordinate motion effect. This work was published in JACS. Isotope
effects are being determined for hydrolysis of cyclis phosphotriesters where phosphorane formation and pseudorotation are involved. The primary O-18 isotope effect for hydrolysis of a triester with an ethylene group in the ring and p-carbamoylphenol as the leaving group is 1.009 at neutral pH. Since this is only 25% of the effect seen with a noncyclic diethyl phosphotriester with the same leaving group, it appears that phosphorane formation, rather than breakdwon, is the major rate limiting step. We are now measuring the secondary O-18 isotope effect. C-13 Isotope: the C-13 isotope effects on human malic enzyme has been determined with results similar to those with the chicken enzyme. Effects in carbamoyl-P for the reaction catalyzed by the ornithine transcarbamoylase that is resistant to an inhibitor that strongly inhibits the normal enzyme. The values drop from 3.5% at low ornithine to 1% at high ornithine, with a half-suppression point of 4mM. Thus the mechanism is not fully
oredered, as is that of the normal enzyme. L-ribulokinase: the kinetic mechanism and substrate specificity of L-ribulokinase have been determined. The mechanism is largely ordered with the sugar adding first, and there is high synergism in the binding of MgATP and sugars (350-fold for L-ribulose). Some randomness is present, however, since L-erythrulose induces competitive substrate inhibition by MgATP that is partial. All 2-ketopentoses are substrates, along with L-arabitol and ribitol, but there is strong preference for the erythro over threo arrangement, and for L over D. For pentitols, only those with the C-2 hydroxyl in D orientation are phosphorylated.
Impacts
Work from this project has led to the development of many kinetic methods for study of enzymatic reactions. Our work with heavy atom isotope effects allows us to determine transition state structures for non-enzymatic as well as enzymatic reactions. Since transition state analogs are often used as medically important drugs, it is important to know what the transition states for enzymatic reactions look like.
Publications
- Substrate specificity and kinetic mechanism of E. coli Ribulokinase. L. V. Lee, B. Gerratana and W. W. Cleland. Arch. Biochem. Biophys. in press (2001)
- Determination of the mechanism of human malic enzyme with natural and alternate dinucleotides by isotope effects. M. Rishavy, A. Yang, L. Tong, and W. W. Cleland. Arch. Biochem. Biophys. in press (2001)
- Hydrolysis of Phosphotriesters: Determination of transition states in parallel reactions by heavy-atom isotope effects. M. Anderson, H. Shim, F. M. Raushel, and W. W. Cleland. J. Am. Chem. Soc. 123, 9245-9253 (2001).
- Mechanistic Roles of Thr134, Tyr160, and Lys164 in the reaction catalyzed by dTDP-Glucose 4.6-Dehydratase. B. Gerratana, W. W. Cleland, and P. A. Frey. Biochemistry 40, 9187-9195 (2001).
- The role of metal ions in catalysis by enolase- an ordered kinetic mechanism for a single substrate enzyme. R. R. Poyner, W. W. Cleland and G. H. Reed. Biochemistry 40, 8009-8017 (2001).
- Characterization of the transition state structure of the reaction on kanamycin nucleotidyltransferase by heavy-atom kinetic isotope effects. B. Gerratana, P. A. Frey and W. W. Cleland. Biochemistry 40, 2972-2977 (2001).
- Regiospecificity assignment for the reaction of kanamycin nucleotidyltransferase from staphlococcus aureus. B. Gerratana, W. W. Cleland and L. A. Reinhardt. Biochemistry 40, 2964-2971 (2001).
- Correlation of low barrier hydrogen bonding and oxyanion binding in transition state analogue complexes of chymotrypsin. D. Neidhart. Y. Wie, C. Cassidy, J. Lin, W. W. Cleland and P. A. Frey. Biochemistry 40, 2439-2447 (2001).
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