Mechanism of nitroalkane oxidase: 2. pH and kinetic isotope effects

Nitroalkane oxidase catalyzes the oxidation of nitroalkanes to aldehydes or ketones with production of nitrite and hydrogen peroxide. pH and kinetic isotope effects with [1, 1-(2)H(2)]nitroethane have been used to study the mechanism of this enzyme. The V/K(ne) pH profile is bell-shaped. A group with a pK(a) value of about 7 must be unprotonated and one with a pK(a) value of 9.5 must be protonated for catalysis. The lower pK(a) value is seen also in the pK(is) profile for the competitive inhibitor valerate, indicating that nitroethane has no significant external commitments to catalysis. The (D)(V/K)(ne) value is pH-independent with a value of 7.5, whereas the (D)V(max) value increases from 1.4 at pH 8.2 to a limiting value of 7.4 below pH 5. The V(max) pH profile decreases at low and high pH, with pK(a) values of 6.6 and 9.5, respectively. Imidazole, which activates the enzyme, affects the V(max) but not the V/K(ne) pH profile. In the presence of imidazole at pH 7 the (D)V(max) value increases to a value close to the intrinsic value, consistent with cleavage of the carbon-hydrogen bond of the substrate being fully rate-limiting for catalysis in the presence of imidazole.     

Analysis of the kinetic isotope effects on initial rates in transient kinetics

A method was described recently for circumventing the difficulties in determining intrinsic kinetic isotope effects from eigenvalues obtained in transient kinetic experiments (Maniscalco, Tally, and Fisher (2004) Arch. Biochem. Biophys. 425, 165-172). The method, based on the isotope effects on initial rates of formation of intermediates, was validated by numerical simulation for only a few linear reaction sequences. A general analytical proof of the validity of the method is given in this work. The mathematical approach, using rate laws and L'H?pital's rule, allows more complex reaction schemes to be analyzed. Several are analyzed in this article, illustrating the broad applicability and possible complications in this approach for determining intrinsic isotope effects. Some possible applications are noted, with particular attention being paid to nonlinear reaction schemes, the effect of measuring signals rather than concentration, and the ability to distinguish stepwise from concerted reactions.     

pH and kinetic isotope effects on the reductive half-reaction of D-amino acid oxidase.

Primary deuterium kinetic isotope and pH effects on the reduction of D-amino acid oxidase by amino acid substrates were determined using steady-state and rapid reaction methods. With D-serine as substrate, reduction of the enzyme-bound FAD requires that a group with a pKa value of 8.7 be unprotonated and that a group with a pKa value of 10.7 be protonated. The DV/Kser value of 4.5 is pH-independent, establishing that these pKa values are intrinsic. The limiting rate of reduction of the enzyme shows a kinetic isotope effect of 4.75, consistent with this as the intrinsic value. At high enzyme concentration (approximately 15 microM) at pH 9,D-serine is slightly sticky (k3/k2 = 0.8), consistent with a decrease in the rate of substrate dissociation. With D-alanine as substrate, the pKa values are perturbed to 8.1 and 11.5. The DV/Kala value increases from 1.3 at pH 9.5 to 5.1 at pH 4, establishing that D-alanine is sticky with a forward commitment of approximately 10. The effect of pH on the DV/Kala value is consistent with a model in which exchange with solvent of the proton from the group with pKa 8.7 is hindered and is catalyzed by H2O and OH- above pH 7 and by H3O+ and H2O below pH 7. With glycine, the pH optimum is shifted to a more basic value, 10.3. The DV/Kgly value increases from 1.26 at pH 6.5 to 3.1 at pH 10.7, consistent with fully reversible CH bond cleavage followed by a pH-dependent step. At pH 10.5, the kinetic isotope effect on the limiting rate of reduction is 3.4.     

pH and deuterium kinetic isotope effects studies on the oxidation of choline to betaine-aldehyde catalyzed by choline oxidase

The FAD-dependent choline oxidase catalyzes the four-electron oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate. The enzyme is capable of accepting either choline or betaine-aldehyde as a substrate, allowing the investigation of the reaction mechanism for both the conversion of choline to betaine-aldehyde and of betaine-aldehyde to glycine-betaine. In the present study, pH and deuterium kinetic isotope effects with [1,2-2H(4)]-choline were used to study the mechanism of oxidation of choline to betaine-aldehyde. The V/K and V(max) pH-profiles increased to limiting values with increasing pH, suggesting the presence of a catalytic base essential for catalysis at the enzyme active site. From the V/K pH-profile with [1,2-2H(4)]-choline, a pK(a) of 8.0 was determined for the catalytic base. This pK(a) was shifted to 7.5 in the V/K pH-profile with choline, indicating a significant commitment to catalysis with this substrate. In agreement with this conclusion, the D(V/K) values decreased from a limiting value of 12.4 below pH 6.5 to a limiting value of 4.1 above pH 9.5. The large D(V/K) values at low pH are consistent with carbon-hydrogen bond cleavage of choline being nearly irreversible and fully rate-limiting at low pH. Based on comparison of amino acid sequences and previous structural and mechanistic studies on other members of the GMC oxidoreductase superfamily, the identity of the catalytic base of choline oxidase is proposed.     

Stereochemistry and kinetic isotope effects in the bovine plasma amine oxidase catalyzed oxidation of dopamine.

The stereochemistry of the bovine plasma amine oxidase catalyzed oxidation of 2-(3,4-dihydroxyphenyl)-ethylamine (domapine) has been investigated by comparing 3H/14C ratios of 3,4-dibenzyloxyphenethyl alcohols, derived from 3,4-dihydroxyphenylacetaldehydes, to starting dopamines chirally labeled at C-1 and C-2. The oxidation of [2RS-3H]-, [2R-3H]-, and [2S-3H]dopamine leads to products which have retained 53, 59, and 47% of their tritium. Similarly, oxidation of [1RS-3H]-, [1R-3H]-, and [1S-3H]dopamine leads to an 80, 80, and 92% retention of tritium. The configurational purity of tritium at C-2 of dopamine and C-1 of the dopamine precursor 3-methoxy-4-hydroxyphenethylamine has been confirmed employing dopamine-beta-hydroxylase (specific for the pro-R hydrogen at C-2) and pea seedling amine oxidase (specific for the pro-S hydrogen at C-1). In addition, chromatographically resolved isozymes of bovine plasma amine oxidase have been demonstrated to lead to the same stereochemical result as pooled enzyme fractions. We have been able to rule out carbon interchange and tritium transfer in the ethylamine side chain of dopamine as the source of the apparent nonstereospecificity. Estimated primary tritium isotope effects are 1 for [2-3H]dopamines and 5--6 and 26--34 for [1R-3H]- and [1S-3H]dopamine, respectively. We propose the presence of alternate dopamine binding modes, characterized by absolute but opposing stereochemistries and differential primary tritium isotope effects at C-1.     

Use of the glucose oxidase system to measure oxygen transfer rates

This investigation used the glucose oxidase system to simulate oxygen transfer rate in fermentation broths. It was demonstrated that the fungal preparation contained sufficient lactonase activity so that D -glucono-δ-lactone did not accumulate and that the rate of production of gluconic acid was proportional to the oxygen uptake rate. Enzyme concentrations of 1.5–2 g/1 were found adequate to determine oxygen absorption rates in shake flasks while maintaining the dissolved oxygen concentration of low levels. The apparent Michaelis constant for oxygen, K_m(O₂), was found to be 27% saturation with air; this value along with experimentally determined uptake rates could be used to calculate dissolved oxygen concentration in lieu of using a dissolved oxygen probe. Enzyme concentrations of 5 g/l were sufficient to give linear acid production and low dissolved oxygen concentrations in a bench-scale fermenter with no foaming or enzyme deactivation. The method is considered more valid and easier to employ than previously utilized techniques such as sulfite oxidation. Extension of the system to evaluating aeration effectiveness and scaleup of fermentation equipment is discussed.     

Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects

The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.     

Alpha-secondary tritium kinetic isotope effects indicate hydrogen tunneling and coupled motion occur in the oxidation of L-malate by NAD-malic enzyme

The NAD-malic enzyme from Ascaris suum catalyzes the divalent metal ion-dependent oxidative decarboxylation of L-malate to give pyruvate and CO2, with NAD+ as the oxidant. Alpha-secondary tritium kinetic isotope effects were measured with NAD+ or APAD+ and L-malate-2-H(D) and several different divalent metal ions. The alpha-secondary tritium kinetic isotope effects are slightly higher than 1 with NAD+ and L-malate as substrates, much larger than the expected inverse isotope effect for a hybridization change from sp2 to sp3. The alpha-secondary tritium kinetic isotope effects are reduced to values near 1 with L-malate-2-D as the substrate, regardless of the metal ion that is used. Data suggest the presence of quantum mechanical tunneling and coupled motion in the malic enzyme reaction when NAD+ and malate are used as substrates. Isotope effects were also measured using the D/T method with NAD+ and Mn2+ as the substrate pair. A Swain-Schaad exponent of 2.2 (less than the value of 3.26 expected for strictly semiclassical behavior) is estimated, suggesting the presence of other slow steps along the reaction pathway. With APAD+ and Mn2+ as the substrate pair, inverse alpha-secondary tritium kinetic isotope effects are observed, and a Swain-Schaad exponent of 3.3 is estimated, consistent with rate-limiting hydride transfer and no quantum mechanical tunneling or coupled motion. Data are discussed in terms of the malic enzyme mechanism and the theory developed by Huskey for D/T isotope effects as an indicator of tunneling [Huskey, W. P. (1991) J. Phys. Org. Chem. 4, 361-366].     

An investigation into the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine using heavy atom kinetic isotope effects

Heavy atom kinetic isotope effects (KIEs) were determined for the butyrylcholinesterase-catalyzed hydrolysis of formylthiocholine (FTC). The leaving-S, carbonyl-C, and carbonyl-O KIEs are ³⁴k = 0.994 ± 0.004, ¹³k = 1.0148 ± 0.0007, and ¹⁸k = 0.999 ± 0.002, respectively. The observed KIEs support a mechanism for both acylation and deacylation where the steps up to and including the formation of the tetrahedral intermediate are at least partially rate determining. These results, in contrast to previous studies with acetylthiocholine, suggest that the decomposition of a tetrahedral intermediate is not rate-determining for FTC hydrolysis. Structural differences between the two substrates are likely responsible for the observed mechanism change with FTC.     

Novel immobilized liposomal glucose oxidase system using the channel protein OmpF and catalase

The reactivity of immobilized glucose oxidase-containing liposomes (IGOL) prepared in our previous work (Wang et al. [2003] Biotechnol Bioeng 83:444-453) was considerably improved here by incorporating the channel protein OmpF from Escherichia coli into the liposome membrane as well as by entrapping inside the liposome's aqueous interior not only glucose oxidase (GO), but also catalase (CA), both from Aspergillus niger. CA was used for decomposing the hydrogen peroxide produced in the glucose oxidation reaction inside the liposomes. The presence of OmpF enhanced the transport of glucose molecules from the exterior of the liposomes to the interior. In a first step of the work, liposomes containing GO and CA (GOCAL) were prepared and characterized. A remarkable protection effect of the liposome membrane on CA inside the liposomes at 40 degrees C was found; the remaining CA activity at 72 h incubation was more than 60% for GOCAL, while less than 20% for free CA. In a second step, OmpF was incorporated into GOCAL membranes, leading to the formation of OmpF-embedded GOCAL (abbreviated GOCAL-OmpF). The activity of GO inside GOCAL-OmpF increased up to 17 times in comparison with that inside GOCAL due to an increased glucose permeation across the liposome bilayer, without any leakage of GO or CA from the liposomes. The optimal system was estimated to contain on average five OmpF molecules per liposome. Finally, GOCAL-OmpF were covalently immobilized into chitosan gel beads. The performance of this novel biocatalyst (IGOCAL-OmpF) was examined by following the change in glucose conversion, as well as by following the remaining GO activity in successive 15-h air oxidations for repeated use at 40 degrees C in an airlift bioreactor. IGOCAL-OmpF showed higher reactivity and reusability than IGOL, as well as IGOL containing OmpF (IGOL-OmpF). The IGOCAL-OmpF gave about 80% of glucose conversion even when the catalyst was used repeatedly four times, while the corresponding conversions were about 60% and 20% for the IGOL and IGOL-OmpF, respectively. Due to the absence of CA, IGOL-OmpF was less stable and resulted in drastically inhibited GO.     

Determination of the intrinsic kinetic constants of immobilized glucose oxidase and catalase

Models of membrane systems containing immobilized glucose oxidase and catalase operating together or independently have been developed. A rotated disk electrode apparatus was employed with novel electrochemical operating conditions to experimentally determine mass transfer and intrinsic kinetic parameters of enzyme-containing membranes. The value of a mass transfer parameter that describes internal and external diffusion was first determined under conditions that do not permit the enzyme reactions. In a subsequent experiment with the reaction allowed, kinetic parameters corresponding to the intrinsic maximal velocity and Michaelis constants of the immobilized enzymes were estimated by regression analysis of data based on an appropriate two- or three- parameter model. It was found that immobilization reduced the maximal intrinsic velocity but had no detectable effect on the Michaelis constants. In all but one case- these methods for membrane characterization are nondestructive and can be used repeatedly on a given membrane. These techniques provide the means for quantitative comparisons of immobilization methods and make possible temporal studies of immobilized enzyme inactivation.     

A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase

Glucose oxidase (GO) and copper amine oxidase (CAO) catalyze the reduction of molecular oxygen to hydrogen peroxide. If a closed-shell cofactor (like FADH(2) in GO and topaquinone (TPQ) in CAO) is electron donor in dioxygen reduction, the formation of a closed-shell species (H(2)O(2)) is a spin forbidden process. Both in GO and CAO, formation of a superoxide ion that leads to the creation of a radical pair is experimentally suggested to be the rate-limiting step in the dioxygen reduction process. The present density functional theory (DFT) studies suggest that in GO, the creation of the radical pair induces a spin transition by spin orbit coupling (SOC) in O(2)(-)(rad), whereas in CAO, it is induced by exchange interaction with the paramagnetic metal ion (Cu(II)). In the rate-limiting step, this spin-transition is suggested to transform the O(2)(-)(rad)-FADH(2)(+)(rad) radical pair in GO and the Cu(II)-TPQ (triplet) species in CAO, from a triplet (T) to a singlet (S) state. For CAO, a mechanism for the O[bond]O cleavage step in the biogenesis of TPQ is also suggested.     

Calculations of intrinsic isotope effects and the detection of tunneling in enzyme-catalyzed reactions

The equation of Northrop [1975, Biochemistry, 14, 2644] for calculating intrinsic isotope effects from observed deuterium and tritium isotope effects of V/K, in which hydrogen is the reference isotope, has been extended to experimental designs using either deuterium or tritium as a reference. Partial derivatives of the intrinsic equations allow calculation of the relative precision of the three referenced isotope effects and these favor the order deuterium > tritium > hydrogen. In comparisons of observed and calculated isotope effects when hydrogen tunneling is present, both the precision and the magnitude of the difference was greater for intrinsic calculations than for exponentiations based upon a breakdown in the Swain-Schaad relationship.     

Mycobacterium tuberculosis beta-ketoacyl-ACP reductase: alpha-secondary kinetic isotope effects and kinetic and equilibrium mechanisms of substrate binding

β-Ketoacyl-ACP reductase catalyzes the NADPH-dependent reduction of β-ketoacyl-acyl carrier protein to generate β-hydroxyacyl-acyl carrier protein and NADP⁺, the second step of the fatty acid elongation system type II of bacteria, plants, and apicomplexan organisms. Here, a modified and more efficient purification protocol is reported for recombinant Mycobacterium tuberculosis β-ketoacyl-ACP reductase (MabA). The increase in α-secondary deuterium kinetic isotope effect values measured at pH 10 as compared to those obtained at pH 7 points to isotope- and pH-sensitive steps occurring concomitantly. Equilibrium and kinetic fluorescence studies demonstrate positive cooperativity in binding of NADPH to MabA, with two forms of free enzyme in solution. Equilibrium dialysis shows no cooperativity in acetoacetyl-CoA binding to the enzyme. Moreover, modest affinity loss occurs when the substrates bind to the monomer as compared to the dimer of MabA. A mechanism of substrate binding to MabA is proposed on the basis of the experimental data.     

Controlling activation of the RNA-dependent protein kinase by siRNAs using site-specific chemical modification

The RNA-dependent protein kinase (PKR) is activated by binding to double-stranded RNA (dsRNA). Activation of PKR by short-interfering RNAs (siRNAs) and stimulation of the innate immune response has been suggested to explain certain off-target effects in some RNA interference experiments. Here we show that PKR's kinase activity is stimulated in vitro 3- to 5-fold by siRNA duplexes with 19 bp and 2 nt 3′-overhangs, whereas the maximum activation observed for poly(I)•poly(C) was 17-fold over background under the same conditions. Directed hydroxyl radical cleavage experiments indicated that siRNA duplexes have at least four different binding sites for PKR's dsRNA binding motifs (dsRBMs). The location of these binding sites suggested specific nucleotide positions in the siRNA sense strand that could be modified with a corresponding loss of PKR binding. Modification at these sites with N²-benzyl-2′-deoxyguanosine (BndG) blocked interaction with PKR's dsRBMs and inhibited activation of PKR by the siRNA. Importantly, modification of an siRNA duplex that greatly reduced PKR activation did not prevent the duplex from lowering mRNA levels of a targeted message by RNA interference in HeLa cells. Thus, these studies demonstrate that specific positions in an siRNA can be rationally modified to prevent interaction with components of cellular dsRNA-regulated pathways.     

The pH dependence of kinetic isotope effects in monoamine oxidase A indicates stabilization of the neutral amine in the enzyme-substrate complex

A common feature of all the proposed mechanisms for monoamine oxidase is the initiation of catalysis with the deprotonated form of the amine substrate in the enzyme-substrate complex. However, recent steady-state kinetic studies on the pH dependence of monoamine oxidase led to the suggestion that it is the protonated form of the amine substrate that binds to the enzyme. To investigate this further, the pH dependence of monoamine oxidase A was characterized by both steady-state and stopped-flow techniques with protiated and deuterated substrates. For all substrates used, there is a macroscopic ionization in the enzyme-substrate complex attributed to a deprotonation event required for optimal catalysis with a pK(a) of 7.4-8.4. In stopped-flow assays, the pH dependence of the kinetic isotope effect decreases from approximately 13 to 8 with increasing pH, leading to assignment of this catalytically important deprotonation to that of the bound amine substrate. The acid limb of the bell-shaped pH profile for the rate of flavin reduction over the substrate binding constant (k(red)/K(s), reporting on ionizations in the free enzyme and/or free substrate) is due to deprotonation of the free substrate, and the alkaline limb is due to unfavourable deprotonation of an unknown group on the enzyme at high pH. The pK(a) of the free amine is above 9.3 for all substrates, and is greatly perturbed (DeltapK(a) approximately 2) on binding to the enzyme active site. This perturbation of the substrate amine pK(a) on binding to the enzyme has been observed with other amine oxidases, and likely identifies a common mechanism for increasing the effective concentration of the neutral form of the substrate in the enzyme-substrate complex, thus enabling efficient functioning of these enzymes at physiologically relevant pH.     

Mechanistic studies of the flavoenzyme tryptophan 2-monooxygenase: deuterium and 15N kinetic isotope effects on alanine oxidation by an L-amino acid oxidase

Tryptophan 2-monooxygenase (TMO) from Pseudomonas savastanoi catalyzes the oxidative decarboxylation of l-tryptophan during the biosynthesis of indoleacetic acid. Structurally and mechanistically, the enzyme is a member of the family of l-amino acid oxidases. Deuterium and 15N kinetic isotope effects were used to probe the chemical mechanism of l-alanine oxidation by TMO. The primary deuterium kinetic isotope effect was pH independent over the pH range 6.5-10, with an average value of 6.0 +/- 0.5, consistent with this being the intrinsic value. The deuterium isotope effect on the rate constant for flavin reduction by alanine was 6.3 +/- 0.9; no intermediate flavin species were observed during flavin reduction. The kcat/Kala value was 1.0145 +/- 0.0007 at pH 8. NMR analyses gave an equilibrium 15N isotope effect for deprotonation of the alanine amino group of 1.0233 +/- 0.0004, allowing calculation of the 15N isotope effect on the CH bond cleavage step of 0.9917 +/- 0.0006. The results are consistent with TMO oxidation of alanine occurring through a hydride transfer mechanism.     

Investigation of the enzymatic mechanism of yeast orotidine-5'-monophosphate decarboxylase using 13C kinetic isotope effects

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).     

Mechanism of Salmonella typhimurium histidinol dehydrogenase: kinetic isotope effects and pH profiles.

L-Histidinol dehydrogenase catalyzes the biosynthetic oxidation of L-histidinol to L-histidine with sequential reduction of two molecules of NAD. Previous isotope exchange results had suggested that the oxidation of histidinol to the intermediate histidinaldehyde occurred 2-3-fold more rapidly than overall catalysis. In this work, we present kinetic isotope effects (KIE) studies at pH 9.0 and at pH 6.7 with stereospecifically mono- and dideuterated histidinols. The data at pH 9.0 support minimal participation of the first hydride transfer and substantial participation of the second hydride transfer in the overall rate limitation. Stopped-flow experiments with protiated histidinol revealed a small burst of NADH production with stoichiometry of 0.12 per subunit, and 0.25 per subunit with dideuterated histidinol, indicating that the overall first half-reaction was not significantly faster than the second reaction sequence. Results from kcat and kcat/KM titrations with histidinol, NAD, and the alternative substrate imidazolyl propanediol demonstrated an essential base with pKa values between 7.7 and 8.4. In KIE experiments performed at pH 6.7 or with a coenzyme analogue at pH 9. 0, the first hydride transfer became more rate limiting. Kinetic simulations based on rate constants estimated from this work fit well with a mechanism that includes a relatively fast, and thermodynamically unfavorable, hydride transfer from histidinol and a slower, irreversible second hydride transfer from a histidinaldehyde derivative. Thus, although the chemistry of the first hydride transfer is fast, both partial reactions participate in the overall rate limitation.     

A method to determine 18 O kinetic isotope effects in the hydrolysis of nucleotide triphosphates

A method to determine 18 O kinetic isotope effects (KIEs) in the hydrolysis of GTP that is generally applicable to reactions involving other nucleotide triphosphates is described. Internal competition, where the substrate of the reaction is a mixture of 18 O-labeled and unlabeled nucleotides, is employed, and the change in relative abundance of the two species in the course of the reaction is used to calculate KIE. The nucleotide labeled with 18 O at sites of mechanistic interest also contains 13C at all carbon positions, whereas the 16 O-labeled nucleotide is depleted of 13C. The relative abundance of the labeled and unlabeled substrates or products is reflected in the carbon isotope ratio (13C/12C) in GTP or GDP, which is determined by the use of a liquid chromatography-coupled isotope ratio mass spectrometer (LC-coupled IRMS). The LC is coupled to the IRMS by an Isolink interface. Carbon isotope ratios can be determined with accuracy and precision greater than 0.04% and are consistent over an order of magnitude in sample amount. KIE values for Ras/NF1(333)-catalyzed hydrolysis of [beta18 O3,13C]GTP were determined by change in the isotope ratio of GTP or GDP or the ratio of the isotope ratio of GDP to that of GTP. KIE values computed in the three ways agree within 0.1%, although the method using the ratio of isotope ratios of GDP and GTP gives superior precision (<0.1%). A single KIE measurement can be conducted in 25 min with less than 5 microg nucleotide reaction product.