Isotope exchange as a probe of the kinetic mechanism of pyrophosphate-dependent phosphofructokinase

Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the bridge position of pyrophosphate to a nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. The maximum rates of isotope exchange at equilibrium for the [14C]fructose 1,6-bisphosphate in equilibrium fructose 6-phosphate, [32P]Pi in equilibrium MgPPi, and Mg[32P]PPi in equilibrium fructose 1,6-bisphosphate exchange reactions increasing all four possible substrate-product pairs in constant ratio are identical, consistent with a rapid equilibrium mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate (F6P)/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate (FBP) pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi, in agreement with initial velocity studies [Bertagnolli, B.L., & Cook, P.F. (1984) Biochemistry 23, 4101]. Neither back-exchange by [32P]Pi nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction.     

Isotope exchange at equilibrium studies with rat muscle adenylosuccinate synthetase

The kinetic mechanism of rat muscle adenylosuccinate synthetase was studied by determining the rates of isotope exchange at equilibrium. A random sequential binding mechanism was indicated for both the forward and reverse reactions. Aspartate, adenylosuccinate, GDP, and Pi were determined to bind in rapid equilibrium. GTP exchanges with both GDP and Pi at the same rate, which is similar to the exchange rate of IMP with adenylosuccinate. Aspartate exchanges with adenylosuccinate at a higher rate than does IMP over the range of concentrations tested. The slower IMP and GTP exchange rates suggest a forward binding mechanism containing a preferred path in which the quaternary complex is most often formed by aspartate binding to the E-GTP-IMP complex. This preferred path is consistent with an interaction between IMP and GTP in the absence of aspartate as determined by isotope scrambling experiments [Bass, M. B., Fromm, H. J., & Rudolph, F. B. (1984) J. Biol. Chem. 259, 12330-12333]. However, the products of such an interaction are tightly bound to the enzyme as no partial exchange reactions between adenylosuccinate and aspartate in the presence or absence of Pi were detected.     

Analysis of the galactosyltransferase reaction by positional isotope exchange and secondary deuterium isotope effects

The mechanism of the galactosyltransferase-catalyzed reaction was probed using positional isotope exchange, alpha-secondary deuterium isotope effects, and inhibition studies with potential transition state analogs. Incubation of [beta-18O2, alpha beta-18O]UDP-galactose and alpha-lactalbumin with galactosyltransferase from bovine milk did not result in any positional isotope exchange. The addition of 4-deoxy-4-fluoroglucose as a dead-end inhibitor did not induce any detectable positional isotope exchange. alpha-Secondary deuterium isotope effects of 1.21 +/- 0.04 on Vmax and 1.05 +/- 0.04 on Vmax/KM were observed for [1-2H]-UDP-galactose. D-Glucono-1,5-lactone, D-galactono-1,4-lactone, D-galactono-1,5-lactone, nojirimycin, and deoxynojirimycin, did not inhibit the galactosyl transfer reaction at concentrations less than 1.0 mM. The magnitude of the secondary deuterium isotope effect supports a mechanism in which the anomeric carbon of the galactosyl moiety has substantial sp2 character in the transition state. Therefore, the cleavage of the bond between the galactose and UDP moieties in the transition state has proceeded to a much greater extent than the formation of the bond between the galactose and the incoming glucose. The lack of a positional isotope exchange reaction indicates that the beta-phosphoryl group of the UDP is not free to rotate in the absence of an acceptor substrate.     

The mechanism of the adenylosuccinate synthetase reaction as studied by positional isotope exchange

In an attempt to gain insight into the mechanism of the rat muscle adenylosuccinate synthetase reaction, experiments using the technique of positional isotope exchange (isotope scrambling) were undertaken. [gamma-18O]GTP was prepared and incubated with Mg2+ and the synthetase in the presence of various ligands. Positional isotope exchange occurred, as measured by nuclear magnetic resonance spectroscopy, when IMP was present. In the absence of IMP, with or without aspartate or succinate, the [gamma-18O]GTP did not exhibit scrambling. These results suggest that the adenylosuccinate synthetase reaction involves the participation of 6-phosphoryl-IMP as an obligatory intermediate. On the basis of experiments carried out in our laboratory as well as in others, we believe the GDP remains bound to the enzyme until the product, adenylosuccinate, is formed. All products may then dissociate randomly from the enzyme. The positional isotope exchange experiments, along with initial-rate experiments carried out in our laboratory, serve to explain the lack of partial exchange reactions associated with the synthetase (Fromm, H. J. (1958) Biochim. Biophys. Acta 29, 255-262), as well as the net inversion of configuration when chiral thio-GTP is converted to thiophosphate (Webb, M. R., Reed, G. H., Cooper, B. F., and Rudolph, F. B. (1984) J. Biol. Chem. 259, 3044-3046).     

Kinetic evidence for the formation of D-alanyl phosphate in the mechanism of D-alanyl-D-alanine ligase

The steady state kinetic mechanism, molecular isotope exchange and the positional isotope exchange (PIX) reactions of D-alanyl-D-alanine ligase from Salmonella typhimurium have been studied. The kinetic mechanism has been determined to be ordered Ter-Ter from initial velocity and product inhibition experiments. The first substrate to bind is ATP followed by the addition of 2 mol of D-alanine. Pi is released, and then D-alanyl-D-alanine and ADP dissociate from the enzyme surface. In the reverse direction D-alanyl-D-alanine exhibits complete substrate inhibition (Ki = 1.15 +/- 0.05 mM) by binding to the enzyme-ATP complex. In the presence of D-alanine, D-alanyl-D-alanine ligase catalyzed the positional exchange of the beta,gamma-bridge oxygen in [gamma-18O4]ATP to a beta-nonbridge position. Two possible alternate dead-end substrate analogs, D-2-chloropropionic acid and isobutyric acid, did not induce a positional isotope exchange in [gamma-18O4]ATP. The positional isotope exchange rate is diminished relative to the net substrate turnover as the concentration of D-alanine is increased. This is consistent with the ordered Ter-Ter mechanism as determined by the steady state kinetic experiments. The ratio of the positional isotope exchange rate relative to the net chemical turnover of substrate (Vex/Vchem) approaches a value of 1.4 as the concentration of D-alanine becomes very small. This ratio is 100 times larger than the ratio of the maximal reverse and forward chemical reaction velocities (V2/V1). This situation is only possible when the reaction mechanism proceeds in two distinct steps and the first step is much faster than the second step. The enzyme was also found to catalyze the molecular isotope exchange of radiolabeled D-alanine with D-alanyl-D-alanine in the presence of phosphate. These results are consistent with the formation of D-alanyl phosphate as a kinetically competent intermediate.     

Application of equilibrium exchange kinetics to studies of kinetic properties at high enzyme concentrations: Experiments with yeast hexokinase P-II

The usefulness of isotope exchange at equilibrium measurements to define both the kinetic parameters and the kinetic reaction mechanism of enzymes at high levels of enzyme is demonstrated by studies of yeast hexokinase P-II. Additional support for this new application of equilibrium exchange kinetics, based upon previously published studies of a variety of enzymes, is also presented, and the advantages and limitations of this approach are discussed.     

Effectors of Escherichia coli aspartate transcarbamoylase differentially perturb aspartate binding rather than the T-R transition

New systematic methods developed for equilibrium isotope exchange kinetics have been used to analyze the effects of activator ATP and inhibitor CTP with Escherichia coli aspartate transcarbamoylase. This indepth approach requires (a) variation of [modifier] with fixed subsaturating levels of substrates, and (b) variation of at least three combinations of reactant-product pairs in constant ratio at equilibrium: [A,B,P,Q], [A,P], and [B,Q] with the co-substrates held constant, in the presence and absence of added modifier. Both ATP and CTP had much stronger effects on the [14C]Asp in equilibrium C-Asp exchange rate than on [32P]C-P in equilibrium Pi. The bisubstrate analog N-phosphonacetyl-L-aspartate activated, then inhibited, Asp in equilibrium C-Asp more strongly than C-P in equilibrium Pi. N-Phosphonacetyl-L-aspartate gave complete (100%) inhibition, whereas CTP inhibition of either exchange was only partial. Substrate saturation curves in the presence and absence of effectors indicate that ATP and CTP perturb the observed values of Rmax and S0.5 in different fashions without appreciably changing the observed Hill number. Computer simulations indicate that the primary site of ATP and CTP action is the association rate for Asp, not the allosteric T-R transition. This finding is substantiated by previous studies in which modified aspartate transcarbamoylase had lost cooperative Asp binding without loss of sensitivity to effectors, or in which sensitivity to one effector could be deleted selectively. The present results, with newly devised computer simulation and analysis methods, illustrate the usefulness of equilibrium isotope exchange kinetic probes for providing unique insights to enzyme regulatory mechanisms, to define exactly which steps are altered in a given kinetic mechanism.     

Kinetic mechanism of orotate phosphoribosyltransferase from Salmonella typhimurium

The chemical mechanism of the phosphoribosyltransferases (PRTases), although largely unknown, may proceed either via a concerted direct-transfer mechanism or with a two-step mechanism involving a carboxonium-like intermediate. To study this question, we have cloned the Salmonella typhimurium pyrE gene, coding for the enzyme orotate phosphoribosyltransferase (EC 2.2.4.10, OPRTase), and developed a bacterial strain that overproduces the enzyme, which we have purified to homogeneity. Initial velocity and product inhibition studies indicated that S. typhimurium OPRTase follows a random sequential kinetic mechanism. This result was further confirmed by equilibrium isotope exchange studies on two substrate-product pairs, PRPP-PPi and OMP-orotate. In addition, the rates of the individual equilibrium isotope exchanges allowed us to conclude that PPi release and PRPP release were the rate-determining steps in the forward and reverse reactions, respectively. Although partial reactions between the two substrate-product pairs, PRPP-PPi and OMP-orotate, were observed, further studies revealed that these exchanges were a result of contaminations. Our results are significant in that S. typhimurium OPRTase, like most PRTases but in contrast to its yeast homologue, follows sequential kinetics. The artifactual partial isotope exchanges found in this work may have implications for similar prior work on the yeast enzyme. In view of the careful isotope effect studies of Parsons and co-workers [Goitein, R.K., Chelsky, D., & Parsons, S.M. (1978) J. Biol. Chem. 253, 2963-2971] and the results obtained by us, we propose that PRTases may involve a direct-transfer mechanism but with low bond order to the leaving pyrophosphate moiety and attacking base.     

Iron isotope fractionation during microbial dissimilatory iron oxide reduction in simulated Archaean seawater

The largest Fe isotope excursion yet measured in marine sedimentary rocks occurs in shales, carbonates, and banded iron formations of Neoarchaean and Paleoproterozoic age. The results of field and laboratory studies suggest a potential role for microbial dissimilatory iron reduction (DIR) in producing this excursion. However, most experimental studies of Fe isotope fractionation during DIR have been conducted in simple geochemical systems, using pure Fe(III) oxide substrates that are not direct analogues to phases likely to have been present in Precambrian marine environments. In this study, Fe isotope fractionation was investigated during microbial reduction of an amorphous Fe(III) oxide-silica coprecipitate in anoxic, high-silica, low-sulphate artificial Archaean seawater at 30 °C to determine if such conditions alter the extent of reduction or isotopic fractionations relative to those observed in simple systems. The Fe(III)-Si coprecipitate was highly reducible (c. 80% reduction) in the presence of excess acetate. The coprecipitate did not undergo phase conversion (e.g. to green rust, magnetite or siderite) during reduction. Iron isotope fractionations suggest that rapid and near-complete isotope exchange took place among all Fe(II) and Fe(III) components, in contrast to previous work on goethite and hematite, where exchange was limited to the outer few atom layers of the substrate. Large quantities of low-δ(56)Fe Fe(II) (aqueous and solid phase) were produced during reduction of the Fe(III)-Si coprecipitate. These findings shed new light on DIR as a mechanism for producing Fe isotope variations observed in Neoarchaean and Paleoproterozoic marine sedimentary rocks.     

The mechanism of glycogen synthetase as determined by deuterium isotope effects and positional isotope exchange experiments

The reaction mechanism for glycogen synthetase from rabbit muscle was examined by alpha-secondary deuterium isotope effects and positional exchange experiments. Incubation of glycogen synthetase with [beta-18O2,alpha beta-18O]UDP-Glc did not result in any detectable positional isotope exchange from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. Glucono-1,5-lactone was found to be a noncompetitive inhibitor versus UDP-Glc. The kinetic constants, K(is) and K(ii), were found to be 91 +/- 4 microM and 0.70 +/- 0.09 mM, respectively. Deoxynojirimycin was a nonlinear inhibitor at pH 7.5. The alpha-secondary deuterium isotope effects were measured with [1-2H]UDP-Glc by the direct comparison method. The isotope effects on Vmax and Vmax/K were found to be 1.23 +/- 0.04 and 1.09 +/- 0.06, respectively. The inhibitory effects by glucono-lactone and deoxynojirimycon plus the large alpha-secondary isotope effect on Vmax have been interpreted to show that an oxocarbonium ion is an intermediate in this reaction mechanism. The lack of a detectable positional isotope exchange reaction in the absence of glycogen suggests the formation of a rigid tight ion pair between UDP and the oxocarbonium ion intermediate.     

Study of ATP-independent stages of reaction catalyzed by phage T4 RNA-ligase

The isotope exchange between [5'-32P]pAP and A(5')ppAp catalyzed by enzyme was shown not to take place in the absence of the acceptor; i. e. the necessity of the acceptor presence during the second step of the process was demonstrated. The isotope exchange reaction between [5'32P]pAp and (pA)5p was studied. It was demonstrated that acceptor (pA)4, slightly whereas the acceptor (pU)4 completely inhibits the isotope reaction. The isotope reaction exchange between [5'-32P]pAp and (pU)4pAp does not take place. The question of existence of adenylated donor elimination mechanism in the presence of "poor" acceptors is considered on the basis of the data obtained.     

Kinetic mechanism and nucleotide specificity of NADH peroxidase

NADH peroxidase is a flavoprotein isolated from Streptococcus faecalis which catalyzes the pyridine nucleotide-dependent reduction of hydrogen peroxide to water. Initial velocity, product, and dead-end inhibition studies have been performed at pH 7.5 and support a ping-pong kinetic mechanism. In the absence of hydrogen peroxide, both transhydrogenation between NADH and thioNAD, and isotope exchange between [14C]NADH and NAD, have been demonstrated, although in both these experiments, the maximal velocity of nucleotide exchange was less than 1.5% the maximal velocity of the peroxidatic reaction. We propose that NADH binds tightly to both oxidized and two-electron reduced enzyme. NADH oxidation proceeds stereospecifically with the transfer of the 4S hydrogen to enzyme, and then, via exchange, to water. No primary tritium kinetic isotope effect was observed, and no statistically significant primary deuterium kinetic isotope effects on V/K were determined, although primary deuterium kinetic isotope effects on V were observed in the presence and absence of sodium acetate. NADH peroxidase thus shares with other flavoprotein reductases striking kinetic, spectroscopic, and stereochemical similarities. On this basis, we propose a chemical mechanism for the peroxide cleaving reaction catalyzed by NADH peroxidase which involves the obligate formation of a flavinperoxide, and peroxo bond cleavage by nucleophilic attack by enzymatic dithiols.     

Isotope ratio determination in boron analysis

Traditionally, boron (B) isotope ratios have been determined using thermal ionization mass spectrometry (TIMS) and, to some extent, secondary ion mass spectrometry (SIMS). Both TIMS and SIMS use a high-resolution mass analyzer, but differ in analyte ionization methods. TIMS uses electrons from a hot filament, whereas SIMS employs an energetic primary ion beam of Ga+, Cs+, or O- for analyte ionization. TIMS can be used in negative or positive ion modes with high sensitivity and precision of B isotope ratio determination. However, isobaric interferences may be a problem, if the sample is not well purified and/or memory of the previous sample is not removed. Time-consuming sample preparation, analyte (B) purification, and sample determination processes limit the applications of TIMS for routine analyses. SIMS can determine B and its isotope ratio in intact solid samples without destroying them, but has poorer resolution and sensitivity than TIMS, and is difficult to standardize for biological samples. Development of plasma-source mass spectrometry (MS) enabled the determination of B concentration and isotope ratio without requiring sample purification. Commonly used plasma-source MS uses an Ar inductively coupled plasma (ICP) as an ionization device interfaced to a low-resolution quadrupole mass analyzer. The quadrupole ICP-MS is less precise than TIMS and SIMS, but is a popular method for B isotope ratio determination because of its speed and convenience. B determination by ICP-MS suffers no spectroscopic interferences. However, sample matrices, memory effects, and some instrument parameters may affect the accuracy and precision of B isotope ratio determination if adequate precautions are not taken. New generations of plasma-source MS instruments using high-resolution mass analyzers provide better sensitivity and precision than the currently used quadrupole ICP-MS. Because of the convenience and high sample throughput, the high-resolution ICP-MS is expected to be the method of choice for B isotope ratio determination. The current state of instrumental capabilities is adequate for B isotope determination. However, precision and accuracy are primarily limited by sample preparation, introduction, and analytical methodology, including 1. Analyte loss and isotope fractionation during sample preparation. 2. The precision of B isotope determination in small samples, especially those containing low concentrations. 3. Difficult matrices. 4. Memory effects. Sample preparation by alkali fusion allows rapid and complete decomposition of hard-to-digest samples, but high-salt environments of the fused materials require extensive sample purification for B ratio determination. The alternative wet-ashing sample decomposition with HF also results in B loss and isotopic fractionation owing to the high volatility of BF3. Open-vessel dry- or wet-ashing methods usually do not work well for animal samples, and are also prone to B loss and contamination. Closed-vessel microwave digestion overcomes these problems, but the digests of biological materials have high C contents, which cause spectral interference on 11B and affect 11B/10B ratios. Exchange separation/preconcentration of B using exchange (cation or anion exchange, B-specific resin, e.g., Amberlite IRA-743) tend to cause B isotope fractionation, and C eluting from these resin columns may interfere with B isotope ratio determination. Memory effects of B that occur during sample determination may cause serious errors in B isotope ratio determination, especially when samples varying in B concentrations and/or isotope composition are analyzed together. Although the utilization of high-resolution plasma-source MS will undoubtedly improve analytical precision, it is the sample preparation, sample introduction, and analytical methodology that represent the primary limitation to accurate and precise B isotope ratio determination.     

Positional isotope exchange and kinetic experiments with Escherichia coli guanosine-5'-monophosphate synthetase

The kinetic mechanism of Escherichia coli guanosine-5'-monophosphate synthetase has been determined by utilizing initial velocity kinetic patterns and positional isotope exchange experiments. The initial velocity patterns of MgATP, XMP, and either NH3 or glutamine (as nitrogen source) were consistent with the ordered addition of MgATP followed by XMP and then NH3. The enzyme catalyzes the exchange of 18O from the beta-nonbridge positions of [beta,beta,beta gamma,gamma,gamma,gamma-18O6]ATP into the alpha beta-bridge position only in the presence of XMP and Mg2+. The exchange reaction did not require NH3. The isotope exchange reaction increased as the XMP concentration increased and then decreased at saturating levels of XMP. These results also support the ordered addition of MgATP followed by XMP. GMP synthetase catalyzes the hydrolysis of ATP to AMP and PPi along with an ATP/PPi exchange reaction in the absence of NH3. These data taken together support a mechanism in which the initial step in the enzymatic reaction involves formation of an adenyl-XMP intermediate. Psicofuranine, an irreversible inhibitor of the enzyme, acts by preventing the release or further reaction of adenyl-XMP with H2O or NH3 but does not suppress the isotope exchange or ATP/PPi exchange reactions. GMP synthetase has also been shown to require a free divalent cation for full activity. When Ca2+ replaces Mg2+ in the reaction, the positional isotope exchange reaction is enhanced but the reaction with NH3 to form GMP is greatly suppressed.     

Improved assay and mechanism of the reaction catalyzed by the chitin synthase from Saccharomyces cerevisiae

1. An improved filtration method is introduced to perform kinetic investigations on the chitin synthase reaction. This method is especially suited for the assay of a large number of samples necessary in enzyme kinetic studies. 2. From initial rate data the possibility could be excluded that the two-substrate reactions occurs by a random or a ping-pong mechanism. 3. Investigations of product inhibition exclude a rapid random mechanism but favour an ordered mechanism with UDP-N-acetylglucosamine as the first substrate. 4. This result was confirmed by isotope exchange studies.     

Steady state and equilibrium exchange kinetic studies of the sheep brain glutamine synthetase reaction.

The kinetic mechanism of the sheep brain glutamine synthetase has been examined by both initial rate kinetics using the glutamate analog beta-glutamate and by isotope exchange measurements at equilibrium. Results of the initial rate studies were compatible with a number of sequential mechanisms but not with a partially or fully ordered rapid equilibrium or a ping-pong mechanism. Kinetic parameters at 37 degrees and pH 7.2 were K beta-Glu = 16 mM, KATP = 0.28 mM, and KNH2OH = 1.4 mM. For all equilibrium exchanges studied (ATP in equilibrium ADP, ATP in equilibrium Pi, and Glu in equilibrium Gln), the rate of exchange rose smoothly to a maximum as all substrates and products were simultaneously raised in a constant ratio. This result is in accord with a random order of substrate addition. A brief treatment of equilibrium exchange rates in cases where all substrate/product pairs are varied together is also presented.     

Mechanistic studies on the bovine liver mitochondrial dihydroorotate dehydrogenase using kinetic deuterium isotope effects

Dihydroorotates deuteriated at both C5 and C6 have been prepared and used to probe the mechanism of the bovine liver mitochondrial dihydroorotate dehydrogenase. Primary deuterium isotope effects on kcat are observed with both (6RS)-[5(S)-2H]- and (6RS)-[6-2H] dihydroorotates (3 and 6, respectively); these effects are maximal at low pH. At pH 6.6, DV = 3.4 for the C5-deuteriated dihydroorotate (3), and DV = 2.3 for the C6-deuteriated compound (6). The isotope effects approach unity at pH 8.8. Analysis of the pH dependence of the isotope effects on kcat reveals a shift in the rate-determining step of the enzyme mechanism as a function of pH. Dihydroorotate oxidation appears to require general base catalysis (pKB = 8.26); this step is completely rate-determining at low pH and isotopically sensitive. Reduction of the cosubstrate, coenzyme Q6, is rate-limiting at high pH and is isotopically insensitive; this step appears to require general acid catalysis (pKA = 8.42). The results of double isotope substitution studies and analysis for substrate isotope exchange with solvent point toward a concerted mechanism for oxidation of dihydroorotate. This finding serves to distinguish further the mammalian dehydrogenase from its parasitic cognate, which catalyzes a stepwise oxidation reaction [Pascal, R., & Walsh, C.T. (1984) Biochemistry 23, 2745].     

Kinetic modeling of hyperpolarized 13C label exchange between pyruvate and lactate in tumor cells

Measurements of the kinetics of hyperpolarized (13)C label exchange between [1-(13)C]pyruvate and lactate in suspensions of intact and lysed murine lymphoma cells, and in cells in which lactate dehydrogenase expression had been modulated by inhibition of the PI3K pathway, were used to determine quantitatively the role of enzyme activity and membrane transport in controlling isotope flux. Both steps were shown to share in the control of isotope flux in these cells. The kinetics of label exchange were well described by a kinetic model that employed rate constants for the lactate dehydrogenase reaction that had been determined previously from steady state kinetic studies. The enzyme showed pyruvate inhibition in steady state kinetic measurements, which the kinetic model predicted should also be observed in the isotope exchange measurements. However, no such pyruvate inhibition was observed in either intact cells or cell lysates and this could be explained by the much higher enzyme concentrations present in the isotope exchange experiments. The kinetic analysis presented here shows how lactate dehydrogenase activity can be determined from the isotope exchange measurements. The kinetic model should be useful for modeling the exchange reaction in vivo, particularly as this technique progresses to the clinic.     

Dihydroorotate dehydrogenase from Clostridium oroticum is a class 1B enzyme and utilizes a concerted mechanism of catalysis

Dihydroorotate dehydrogenase from Clostridium oroticum was purified to apparent homogeneity and found to be a heterotetramer consisting of two alpha (32 kDa) and two beta (28 kDa) polypeptides. This subunit composition, coupled with known cofactor requirements and the ability to transfer electrons from L-dihydroorotate to NAD(+), defines the C. oroticum enzyme as a family 1B dihydroorotate dehydrogenase. The results of steady-state kinetic analyses and isotope exchange studies suggest that this enzyme utilizes a ping-pong steady-state kinetic mechanism. The pH-k(cat) profile is bell-shaped with a pK(a) of 6.4 +/- 0.1 for the ascending limb and 8. 9 +/- 0.1 for the descending limb; the pH-k(cat)/K(m) profile is similar but somewhat more complex. The pK(a) values of 6.4 and 8.9 are likely to represent the ionizations of cysteine and lysine residues in the active site which act as a general base and an electrostatic catalyst, respectively. At saturating levels of NAD(+), the isotope effects on (D)V and (D)(V/K(DHO)), obtained upon deuteration at both the C(5)-proR and C(5)-proS positions of L-dihydroorotate, increase from a value of unity at pH >9.0 to sizable values at low pH due to a high commitment to catalysis at high pH. At pH = 6.5, the magnitude of the double isotope effects (D)V and (D)(V/K(DHO)), obtained upon additional deuteration at C(6), is consistent with a mechanism in which C(5)-proS proton transfer and C(6)-hydride transfer occur in a single, partially rate-limiting step.     

Determination of the rate-limiting steps and chemical mechanism of fructokinase by isotope exchange, isotope partitioning, and pH studies.

Isotope exchange studies show that beef liver fructokinase has a random kinetic mechanism in which release of fructose from the enzyme is slower than that catalytic reaction. The stickiness of fructose in the presence of MgATP is confirmed by isotope partition studies, which show it to be released 0.53 times as fast as V1/Et in the presence, and 80--130 times as fast in the absence of MgATP. Fructose-1-P release from it binary complex is not at all rate limiting in the forward direction since no exchange of MgADP back into MgATP could be observed during the forward reaction. Failure to find any isotope effect by the equilibrium perturbation method with [1-18O]fructose (upper limit, 1.003, shows that P--O bond cleavage or formation is not rate limiting. The pH profiles for the forward reaction show a group (probably carboxyl with pK 5.7-6.0 and deltaHion = 0) that must be ionized and a group (perhaps lysine, with pK 9--10, and deltaHion 5-9 kcal/mol) which must be protonated for activity. The profile for the back reaction shows only a group with pK 5.5--6 that must be protonated for activity. A chemical mechanism is proposed in which a carboxyl group on the enzyme accepts a proton from the 1-hydroxyl of fructose during the forward reaction and donates it back during the reverse reaction.