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1.
Arch Biochem Biophys ; 635: 87-95, 2017 12 01.
Article in English | MEDLINE | ID: mdl-29111294

ABSTRACT

Binding isotope effects (BIEs) associated with binding of four triazole-based ligands to HIV-1 reverse transcriptase have been calculated at the QM/MM MD level of theory. Two main binding sites: allosteric cavity and RNase H active site, as well as three other sites reported in the literature (the Knuckles, the NNRTI Adjacent, and Incoming Nucleotide Binding) have been considered. The interactions between inhibitors and these protein sites have been quantified by binding free energies obtained from free energy perturbation (FEP) calculations, supported by interaction energy analysis. It has been shown that binding in the allosteric cavity can be distinguished from binding to other sites based on BIEs as it is associated with normal 18O-BIEs of the carbonyl oxygen atom while binding to RNase H active site is characterized by inverse binding isotope effect (18O-BIE < 1). For other sites 18O-BIEs close to unity are predicted. This information points to oxygen binding isotope effects of carbonyl group as indicative of the actual binding site of studied inhibitors.


Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , Models, Chemical , Molecular Docking Simulation , Oxygen Isotopes/chemistry , Triazoles/chemistry , Binding Sites , Computer Simulation , Protein Binding
2.
Phys Chem Chem Phys ; 18(1): 310-7, 2016 Jan 07.
Article in English | MEDLINE | ID: mdl-26612030

ABSTRACT

Understanding of protein-ligand interactions is crucial for rational drug design. Binding isotope effects, BIEs, can provide intimate details of specific interactions between individual atoms of an inhibitor and the binding pocket. We have applied multi-scale QM/MM simulations to evaluate binding energetics of a novel triazole-based non-nucleoside inhibitor of HIV-1 reverse transcriptase and to calculate associated BIEs. The binding sites can be distinguished based on the (18)O-BIE.


Subject(s)
HIV Reverse Transcriptase/antagonists & inhibitors , HIV Reverse Transcriptase/chemistry , Quantum Theory , Reverse Transcriptase Inhibitors/pharmacology , Thermodynamics , Triazoles/pharmacology , Binding Sites , HIV Reverse Transcriptase/metabolism , Models, Molecular , Oxygen Isotopes , Reverse Transcriptase Inhibitors/chemistry , Structure-Activity Relationship , Triazoles/chemistry
3.
J Org Chem ; 70(10): 4022-7, 2005 May 13.
Article in English | MEDLINE | ID: mdl-15876091

ABSTRACT

[reaction: see text] The transition structures and alpha-carbon 12C/13C kinetic isotope effects for 22 S(N)2 reactions between methyl chloride and a wide variety of nucleophiles have been calculated using the B1LYP/aug-cc-pVDZ level of theory. Anionic, neutral, and radical anion nucleophiles were used to give a wide range of S(N)2 transition states so the relationship between the magnitude of the alpha-carbon kinetic isotope effect and transition-state structure could be determined. The results suggest that the alpha-carbon 12C/13C kinetic isotope effects for S(N)2 reactions will be large (near the experimental maximum) and that the curve relating the magnitude of the KIE to the percent transfer of the alpha-carbon from the nucleophile to the leaving group in the transition state has a broad maximum. This means very similar KIEs will be found for early, symmetric, and late transition states and that one cannot use the magnitude of these KIEs to estimate transition-state structure.

4.
J Inorg Biochem ; 86(4): 681-9, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11583786

ABSTRACT

We have calculated hydrogen kinetic isotope effects (KIEs) for the first step of the methylmalonyl-CoA mutase reaction, including multidimensional tunneling correction at the zero curvature (ZCT) level, and compared them with the experimental values. Both alternative mechanisms of this step, concerted and stepwise, can be accommodated. It turned out to be essential to include Arg207 hydrogen-bonded to the reactant in the mechanism predicting simultaneous breaking of the Co-C bond of AdoCbl and hydrogen atom transfer. The consequence of the stepwise mechanism is a much larger facilitation of the homolytic dissociation of the carbon-cobalt bond by the enzyme than currently appreciated; our results suggest lowering of the activation energy by about 23 kcal mol(-1). We have also shown that large hydrogen KIEs of tunneling origin do not necessarily break the Swain-Schaad equation. Furthermore, when this equation does not hold, the exponent may be smaller in the presence of tunneling than it is at the semi-classical limit, indicating that nonclassical behavior may be a more common phenomenon than expected.


Subject(s)
Methylmalonyl-CoA Mutase/chemistry , Methylmalonyl-CoA Mutase/metabolism , Deuterium , Hydrogen/chemistry , Hydrogen Bonding , Kinetics , Models, Chemical , Thermodynamics , Tritium
6.
J Org Chem ; 66(16): 5534-6, 2001 Aug 10.
Article in English | MEDLINE | ID: mdl-11485479

ABSTRACT

Nitrogen kinetic isotope effects on the decarboxylation of 4-pyridylacetic acid have been measured in solvents of different polarity and have been found to vary from the inverse value of 0.994 to the normal value of 1.002 upon increase of water content of the binary dioxane--water solvent from 25% to 75% (v/v), respectively. These changes were successfully modeled theoretically and shown to originate from the large inverse nitrogen isotope effect on the equilibrium between acidic and zwitterionic forms.

7.
J Am Chem Soc ; 123(31): 7683-6, 2001 Aug 08.
Article in English | MEDLINE | ID: mdl-11480991

ABSTRACT

The rate constants and kinetic isotope effects for decarboxylation of 4-pyridylacetic acid depend strongly on whether the solvent is water or dioxane, and the present paper interprets this finding. We calculate the solvent dependence of the free energy barrier and of the (13)C and (18)O kinetic isotope effects using a quantum mechanical solvation model based on class IV charges and semiempirical atomic surface tensions. The calculations provide a consistent interpretation of the experimental results, which provides a striking confirmation of the soundness of the solvation modeling. Even more significantly, the agreement of theory and experiment gives us confidence in the physical picture of the reaction provided by the model. This indicates that the location of the transition state, as measured by the length of the breaking C--C bond, is 0.24 A later than the gas phase in dioxane and 0.37 A later than the gas phase in water. Charge development at the transition state also depends strongly on the solvent; in particular the CO(2) moiety is 0.07 electronic charge units more negative at the transition state in dioxane than in water.

8.
J Am Chem Soc ; 123(19): 4550-5, 2001 May 16.
Article in English | MEDLINE | ID: mdl-11457241

ABSTRACT

We have found chlorine kinetic isotope effects on the dehalogenation catalyzed by haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 to be 1.0045 +/- 0.0004 for 1,2-dichloroethane and 1.0066 +/- 0.0004 for 1-chlorobutane. The latter isotope effect approaches the intrinsic chlorine kinetic isotope effect for the dehalogenation step. The intrinsic isotope effect has been modeled using semiempirical and DFT theory levels using the ONIOM QM/QM scheme. Our results indicate that the dehalogenation step is reversible; the overall irreversibility of the enzyme-catalyzed reaction is brought about by a step following the dehalogenation.


Subject(s)
Chlorine/chemistry , Hydrolases/chemistry , Algorithms , Butanes/chemistry , Ethylene Dichlorides/chemistry , Isotopes , Kinetics , Models, Chemical , Models, Molecular , Xanthobacter/enzymology
9.
J Pineal Res ; 29(2): 125-7, 2000 Sep.
Article in English | MEDLINE | ID: mdl-10981826

ABSTRACT

Theoretical calculations of the HO* radical reaction with the melatonin molecule were performed. Reaction pathways with C2, C3, C4, C6 and C7 as the target carbon atoms and corresponding radical adducts were studied. Low activation energies of all adducts suggest that these reactions should occur quite easily and with rather low selectivity. C2 carbon as the most probable site of attack and C3 as the least probable one are proposed.


Subject(s)
Computer Simulation , Hydroxyl Radical/chemistry , Melatonin/chemistry , Models, Chemical , Carbon/chemistry , Thermodynamics
10.
Arch Biochem Biophys ; 370(2): 216-21, 1999 Oct 15.
Article in English | MEDLINE | ID: mdl-10510280

ABSTRACT

The mechanism by which phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) catalyzes the reversible elimination of ammonia from phenylalanine yielding (E)-cinnamic acid has gained much attention in the recent years. Dehydroalanine is essential for the catalysis. It was assumed that this prostetic group acts as the electrophile, leading to a covalently bonded enzyme-intermediate complex with quarternary nitrogen of phenylalanine. Recently, an alternative mechanism has been suggested in which the enzyme-intermediate complex is formed in a Friedel-Crafts reaction between dehydroalanine and orthocarbon of the aromatic ring. Using semiempirical calculations we have shown that these two alternative mechanisms can be distinguished on the basis of the hydrogen secondary kinetic isotope effect when tritium label is placed in the orthopositions. Our calculations indicated also that the kinetic isotope effect measured using ring-labeled d(5)-phenylalanine could not be used to differentiate these alternative mechanisms. Measured secondary tritium kinetic isotope effect shows strong dependence on the reaction progress, starting at the inverse value of k(H)/k(T) = 0.85 for 5% conversion and reaching the normal value of about 1.15 as the conversion increases to 20%. This dependence has been interpreted in terms of a complex mechanism with initial formation of the Friedel-Crafts type intermediate.


Subject(s)
Phenylalanine Ammonia-Lyase/metabolism , Alanine/analogs & derivatives , Alanine/chemistry , Catalytic Domain , Deuterium , Kinetics , Models, Chemical , Phenylalanine Ammonia-Lyase/chemistry , Rhodotorula/enzymology , Substrate Specificity , Tritium
11.
Anal Chem ; 70(17): 3548-52, 1998 Sep 01.
Article in English | MEDLINE | ID: mdl-21644703

ABSTRACT

Two methods have been used to measure the chlorine leaving group kinetic isotope effect for the S(N)2 reduction of benzyl chloride to toluene by sodium borohydride in DMSO at 30.000 °C. The reaction was monitored by titrating the unreacted borohydride ion. One method involved determining the chlorine isotope effect using the classical IRMS method, which requires the conversion of the chloride ions into gaseous methyl chloride that is analyzed in an isotope ratio mass spectrometric analyses (Hill, J. W.; Fry, A. J. Am. Chem. Soc. 1962, 84, 2763. Taylor, J. W.; Grimsrud, E. P. Anal. Chem. 1969, 41, 805.). Two different measurements using this method yielded isotope effects of k(35)/k(37) = 1.007 19 ± 0.000 19 and 1.007 64 ± 0.000 19. The second method was a new technique where the ratio of the chlorine isotopes was obtained by fast atom bombardment mass spectrometry on the silver chloride recovered from the reaction, i.e., from the first step in the classical procedure. Therefore, the new method is much simpler and avoids the time-consuming preparation, purification, and recovery of the gaseous methyl chloride. Although the experimental error is larger (k(35)/k(37) = 1.008 03 ± 0.00 10 and 1.008 02 ± 0.000 65) when the new technique is used to analyze the silver chloride samples from the same set of experiments that were used to measure the isotope effect by the classical method, the chlorine isotope effect found by the two methods is identical within experimental error. This large chlorine kinetic isotope effect indicates there is considerable C(α)-Cl bond rupture in the S(N)2 transition state.

12.
J Org Chem ; 62(21): 7305-7309, 1997 Oct 17.
Article in English | MEDLINE | ID: mdl-11671844

ABSTRACT

Nitrogen and carbon kinetic isotope effects were measured on the decarboxylation of 3-carboxybenzisoxazole at room temperature. The nitrogen isotope effect in acetone is 1.0312 +/- 0.0006. The carbon isotope effect shows no dependence on the solvent polarity: 1.0448 +/- 0.0007 in 1,4-dioxane, 1.0445 +/- 0.0001 in acetonitrile, 1.0472 +/- 0.0013 in DMF, and 1.0418 +/- 0.0027 in water. These isotope effects were modeled theoretically at the semiempirical (AM1, PM3, SAM1) and ab initio (up to B3LYP/6-31++G) levels. The comparison of the theoretical and experimental results leads to the conclusion that none of the theory levels employed is capable of quantitatively predicting these isotope effects.

13.
Comput Chem ; 19(3): 231-40, 1995 Sep.
Article in English | MEDLINE | ID: mdl-7551556

ABSTRACT

An overview of calculations of isotope effects on biochemical and chemical reactions using quantum chemistry methods is presented . Usefulness of different levels of theoretical scrutiny for such calculations is critically discussed.


Subject(s)
Isotopes , Chemical Phenomena , Chemistry, Physical , Decarboxylation , Isoxazoles/metabolism , Kinetics , Methyltransferases , Models, Chemical , Molecular Structure , Nitriles/metabolism , Protein Binding
14.
Biochemistry ; 34(18): 6050-8, 1995 May 09.
Article in English | MEDLINE | ID: mdl-7742308

ABSTRACT

An equilibrium isotope effect on association of [1-18O]oxamate to form a ternary complex with lactate dehydrogenase and NADH of 0.9840 +/- 0.0027 has been measured by equilibrium dialysis and whole molecule isotope ratio mass spectrometry. Semiempirical calculations of vibrational frequencies using various models for solvent were shown to predict an inverse equilibrium isotope effect on association. However, the calculated effect cannot be directly attributed to one specific normal mode or vibrational force constant. Analysis of the carboxylate interaction with the guanidinium ion showed that the ionic interaction increased the torsional force constant for rotation about the carbon-carbon bond of oxamate. The minimum energy geometry for oxamate interacting with methyl guanidinium predicts that the plane of the oxamate carboxylate will be at an oblique angle to the plane defined by the guanidinium nitrogens. The combination of experimental and calculated equilibrium isotope effects on association holds the potential to improve the characterization of the interaction of ligands with protein active sites.


Subject(s)
L-Lactate Dehydrogenase/chemistry , NAD/chemistry , Oxamic Acid/chemistry , Models, Theoretical , Molecular Conformation , Oxygen Isotopes
15.
Comput Chem ; 19(1): 11-20, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7735700

ABSTRACT

An overview of calculations of isotope effects on biochemical and chemical reactions using quantum chemistry methods is presented. Usefulness of different levels of theoretical scrutiny for such calculations is critically discussed.


Subject(s)
Isotopes , Models, Chemical , Computer Simulation , Decarboxylation , Enzymes/metabolism , Hydrolysis , In Vitro Techniques , Kinetics , Molecular Structure , Quantum Theory
16.
Biochemistry ; 34(8): 2577-83, 1995 Feb 28.
Article in English | MEDLINE | ID: mdl-7873538

ABSTRACT

Kinetic isotope effects on association have been measured using the remote label methodology developed by O'Leary and Marlier (1979). The isotope effect on V/KA for the first substrate in an obligatorily ordered mechanism is an isotope effect on its second-order rate constant for association with the enzyme. With phosphoenolpyruvate carboxylase the 18(V/KPEP) when the bridging O is labeled decreases from 1.0056 +/- 0.0007 to 0.9943 +/- 0.0002 as the concentration of bicarbonate, the second substrate, increases from 2 to 200 mM. With pyruvate kinase the 18(V/KPEP) decreases from 1.0024 +/- 0.0014 to 0.9928 +/- 0.0027 as the concentration of ADP increases from 1.5 to 30 mM. These inverse kinetic isotope effects are best understood as arising from an isotope effect on the rate constant for forming the Michaelis complex of enzyme and substrate. The inverse value suggests that the bridging oxygen is in a vibrationally stiffer environment in the transition state for the association reaction.


Subject(s)
Phosphoenolpyruvate Carboxylase/metabolism , Phosphoenolpyruvate/metabolism , Pyruvate Kinase/metabolism , Animals , Binding Sites , Carbon Isotopes , In Vitro Techniques , Kinetics , Models, Chemical , Oxygen Isotopes , Phosphoenolpyruvate/chemistry , Rabbits , Substrate Specificity
17.
Eur Biophys J ; 23(5): 353-60, 1994.
Article in English | MEDLINE | ID: mdl-7835319

ABSTRACT

Semiempirical methods have been used in an attempt to predict theoretically the experimentally observed value of 0.9840 for the oxygen isotope effect on binding of oxamate to lactate dehydrogenase. The overall strategy involved vibrational analysis of oxamate in two different environments; that of the active site residues and in aqueous solution. The comparison of calculated values with the experimentally determined isotope effect proved the AM1 Hamiltonian to be superior to the PM3 Hamiltonian in this modelling. While most tested methods of accounting for solvent effects on the vibrational frequencies of the solute yielded similar results it turned out that what was crucial for the purpose of determination of the isotope effect was the model of oxamate in the active site of the enzyme. In particular, the major factor responsible for the inverse value of this isotope effect can be ascribed to the formation of an ordered, bifurcated hydrogen bond between the oxamate carboxylate and the guanidinium group of the active site histidine.


Subject(s)
L-Lactate Dehydrogenase/chemistry , L-Lactate Dehydrogenase/metabolism , Oxamic Acid/metabolism , Oxygen Isotopes , Oxygen , Animals , Binding Sites , Dogfish , Hydrogen Bonding , Models, Molecular , NAD/metabolism , Protein Binding , Protein Conformation
18.
Biochemistry ; 30(25): 6216-23, 1991 Jun 25.
Article in English | MEDLINE | ID: mdl-2059628

ABSTRACT

Orotidine-5'-monophosphate decarboxylase (ODCase) from Saccharomyces cerevisiae displays an observed 13C kinetic isotope effect of 1.0247 +/- 0.0008 at 25 degrees C, pH 6.8. The observed isotope effect is sensitive to changes in the reaction medium, such as pH, temperature, or glycerol content. The value of 1.0494 +/- 0.0006 measured at pH 4.0, 25 degrees C, is not altered significantly by temperature or glycerol, and thus the intrinsic isotope effect for the reaction is apparently being observed under these conditions and decarboxylation is almost entirely rate-determining. These data require a catalytic mechanism with freely reversible binding and one in which a very limited contribution to the overall rate is made by chemical steps preceding decarboxylation; the zwitterion mechanism of Beak and Siegel [Beak, P. & Siegel, B. (1976) J. Am. Chem. Soc. 98, 3601-3606], which involves only protonation of the pyrimidine ring, is such a mechanism. With use of an intrinsic isotope effect of 1.05, a partitioning factor of less than unity is calculated for ODCase at pH 6.0, 25 degrees C. A quantitative kinetic analysis using this result excludes the possibility of an enzymatic mechanism involving covalent attachment of an enzyme nucleophile to C-5 of the pyrimidine ring. The observed isotope effect does not rise to the intrinsic value above pH 8.5; instead, the observed isotope effects at 25 degrees C plotted against pH yield an asymmetric curve that at high pH plateaus at about 1.035. These data, in conjunction with the pH profile of Vmax/km, fit a kinetic model in which an enzyme proton necessary for catalysis is titrated at high pH, thus providing evidence for the catalytic mechanism of Beak and Siegel (1976).


Subject(s)
Orotidine-5'-Phosphate Decarboxylase/metabolism , Saccharomyces cerevisiae/enzymology , Carbon Isotopes , Glycerol/metabolism , Hydrogen-Ion Concentration , Kinetics , Temperature
19.
Photosynth Res ; 28(2): 69-76, 1991 May.
Article in English | MEDLINE | ID: mdl-24414860

ABSTRACT

Structural analogues of the NADP(+) were studied as potential coenzymes and inhibitors for NADP(+) dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N(6)-etheno-nicotinamide adenine dinucleotide phosphate (∈ NADP(+)), 3-acetylpyridine-adenine dinucleotide phosphate (APADP(+)), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP(+)) and ß-nicotinamide adenine dinucleotide 2': 3'-cyclic monophosphate (2'3'NADPc(+)) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in Vmax for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP(+)), 3-aminopyridine-adenine dinucleotide phosphate (AADP(+)), adenosine 2'-monophosphate (2'AMP) and adenosine 2': 3'-cyclic monophosphate (2'3'AMPc) were competitive inhibitors with respect to NADP(+), while ß-nicotinamide adenine dinucleotide 3'-phosphate (3'NADP(+)), NAD(+), adenosine 3'-monophosphate (3'AMP), adenosine 2': 5'-cyclic monophosphate (2'5'AMPc), 5'AMP, 5'ADP, 5'ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2'-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C4 atom of the pyridine ring is the major factor that governs the coenzyme activity.

20.
Talanta ; 34(10): 877-83, 1987 Oct.
Article in English | MEDLINE | ID: mdl-18964423

ABSTRACT

Measurements of the kinetic isotope effects of heavy atoms are discussed from an analytical point of view. The discussion includes the choice of isotope, the procedure for measurement, and methods for increasing the kinetic isotope effect. The procedures most commonly used and newly developed techniques are presented and their advantages and disadvantages considered.

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