Kinetic deuterium isotope effects on the N-demethylation of tertiary amides by cytochrome P-450

Liver microsomal cytochrome P-450 readily N-dealkylates N,N-dimethylamides. N-Methyl-N-hydroxymethyl amides were isolated as intermediates and characterized by gas chromatography-mass spectrometry as their trimethylsilyl ethers. Intramolecular kinetic deuterium isotope effects measured for the enzymic N-demethylation of a series of 12 aromatic and aliphatic N-methyl-N-trideuteriomethyl amides, RCON(CH3)CD3, varied from 3.6 to 6.9 but were independent of both amide bond rotation rate and substrate oxidation potential. These values, which represent a lower limit to the intrinsic isotope effect (Dkintrinsic), are significantly larger than those observed for anodic N-demethylation and are consistent with a mechanism involving hydrogen atom abstraction. On the other hand, with N,N-dimethylbenzamide the intermolecular kinetic deuterium isotope effects on Vmax and Vmax/Km were found to be much smaller (1.23 and 1.75, respectively) indicating substantial suppression of the intrinsic isotope effect. Such suppression indicates the occurrence of a rate-limiting step other than the isotopically sensitive step together with a strong commitment to catalysis.     

Oxygen-18 studies on the oxidative deamination mechanism of alicyclic primary amines in rabbit liver microsomes

The mechanism of microsomal oxidative deamination of alicyclic primary amines: cyclopentylamine, cyclohexylamine, cycloheptylamine, 1- and 2-aminoindan, 1- and 2-aminotetralin, was studied under an atmosphere of ¹⁸O₂ or in a medium containing H₂¹⁸O. The oxygen-18 contents of the products determined by gas-liquid chromatography/mass spectrometry revealed that almost all (75–100 atom%) of the oxygen of oximes was derived from molecular oxygen, whereas a part (4–25 atom% ) of the oxygen of ketones. The studies on the hydrolysis of oximes and the oxygen exchange reaction of ketones proved that the latter proceeded at a considerable rate ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 9.5–336}\text{min}$) and the former made a minor contribution, to explain why the major portion (75–96 atom%) of the oxygen in ketones was derived from water. The results support the mechanism that microsomal deamination proceeds mainly through a carbinolamine intermediate, which is initially hydroxylated at the α carbon to the amino group, partially equilibrating with the imine, and then rearranges to form a ketone and ammonia.     

Kinetic deuterium isotope effects for 7-alkoxycoumarin O-dealkylation reactions catalyzed by human cytochromes P450 and in liver microsomes. Rate-limiting C-H bond breaking in cytochrome P450 1A2 substrate oxidation

7-Ethoxy (OEt) coumarin has been used as a model substrate in many cytochrome P450 (P450) studies, including the use of kinetic isotope effects to probe facets of P450 kinetics. P450s 1A2 and 2E1 are known to be the major catalysts of 7-OEt coumarin O-deethylation in human liver microsomes. Human P450 1A2 also catalyzed 3-hydroxylation of 7-methoxy (OMe) coumarin at appreciable rates but P450 2E1 did not. Intramolecular kinetic isotope effects were used as estimates of the intrinsic kinetic deuterium isotope effects for both 7-OMe and 7-OEt coumarin dealkylation reactions. The apparent intrinsic isotope effect for P450 1A2 (9.4 for O-demethylation, 6.1 for O-deethylation) showed little attenuation in other competitive and noncompetitive experiments. With P450 2E1, the intrinsic isotope effect (9.6 for O-demethylation, 6.1 for O-deethylation) was attenuated in the noncompetitive intermolecular experiments. High noncompetitive intermolecular kinetic isotope effects were seen for 7-OEt coumarin O-deethylation in a baculovirus-based microsomal system and five samples of human liver microsomes (7.3-8.1 for O-deethylation), consistent with the view that P450 1A2 is the most efficient P450 catalyzing this reaction in human liver microsomes and indicating that the C-H bond-breaking step makes a major contribution to the rate of this P450 (1A2) reaction. Thus, the rate-limiting step appears to be the chemistry of the breaking of this bond by the activated iron-oxygen complex, as opposed to steps involved in the generation of the reactive complex. The conclusion about the rate-limiting step applies to all of the systems studied with this model P450 1A2 reaction including human liver microsomes, the most physiologically relevant.     

Carbon-13 and deuterium isotope effects on oxalacetate decarboxylation by pyruvate carboxylase

Deuterium and 13C isotope effects for the enzymic decarboxylation of oxalacetate showed that both deuterium- and 13C-sensitive steps in the reaction are partially rate limiting. A normal alpha-secondary effect of 1.2 per deuterium was calculated for the reaction in which pyruvate-d3 was the substrate, suggesting that the enolate of pyruvate was an intermediate in the reaction. The large normal alpha-secondary deuterium isotope effect of 1.7 when oxalacetate-d2 was the substrate suggests that the motions of the secondary hydrogens are coupled to that of the primary hydrogen during the protonation of the enolate of pyruvate. The reduction in the magnitude of the 13C isotope effect for the oxamate-dependent decarboxylation of oxalacetate from 1.0238 to 1.0155 when the reaction was performed in D2O (primary deuterum isotope effect = 2.1) clearly indicates that the transfer of the proton and carboxyl group between biotin and pyruvate does not occur via a single concerted reaction. Mechanisms in which biotin is activated to react with CO2 (prior to transfer of the proton on N-1) by bond formation between the sulfur and the ureido carbon, or in which the sequence of events is decarboxylation of oxalacetate, proton transfer from biotin to enolpyruvate, and carboxylation of enolbiotin, predict that the 13C isotope effect in D2O should be substantially lower than the observed value. A stepwise mechanism that does fit the data is one in which a proton is removed from biotin by a sulfhydryl group on the enzyme prior to carboxyl transfer, as long as the sulfhydryl group has an abnormally low pK.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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 isotope effects on cytochrome P-450-catalyzed oxidation reactions: full expression of the intrinsic isotope effect during the O-deethylation of 7-ethoxycoumarin by liver microsomes from 3-methylcholanthrene-induced hamsters

The intrinsic primary deuterium isotope effect for the O-deethylation of 7-ethoxycoumarin has been estimated by the Northrop method [D. B. Northrop (1977) in Isotope Effects on Enzyme-Catalyzed Reactions (Cleland, W. W., O'Leary, M. H., and Northrop, D. B., eds.), pp. 122-152, University Park Press, Baltimore] for the microsomal cytochrome P-448 system from 3-methylcholanthrene-induced hamster livers. The intrinsic isotope effect (Dk = 5.5) was found to be equivalent to the observed deuterium isotope effect, demonstrating that the isotope effect for this reaction was fully expressed by this cytochrome P-448 system. These data unequivocally demonstrate that C-H bond cleavage is the rate-limiting step in the overall reaction catalyzed by this system. The decrease in the rate of product formation, occurring as a consequence of deuterium substitution, resulted in a reduction in the quantity of substrate metabolized but was not accompanied by the change in regiospecificity observed in previous studies with a hepatic cytochrome P-448 isozyme purified from 3-methylcholanthrene-induced rats. These data demonstrate that the catalytic site of the hamster isozyme(s) offers more constraints to 7-ethoxycoumarin reorientation than does the catalytic site of the rat liver isozyme.     

Mechanism of urocanase as studied by deuterium isotope effects and labeling patterns

Nicotinamide adenine dinucleotide (NAD) dependent urocanase (4'-imidazolone-5'-propionate hydro-lyase, EC 4.2.1.49) from Pseudomonas putida was found to catalyze an exchange reaction between solvent and the 4'-hydrogen of urocanate or imidazolepropionate at a rate faster than that of overall deuterium was compared to unlabeled urocanate as a substrate, no isotope rate effect was noted. For examination of the possibility of an NAD+-mediated intramolecular hydride transfer of the 4'-hydrogen to a position on the side chain of oxoimidazolepropionate, the origins of hydrogen at positions 2 and 3 in the propionate chain were studied as a function of reaction time and extent of exchange of the 4'-hydrogen. No transfer of hydrogen from the 4' position to the side chain was observed, thereby eliminating mechanisms requiring hydride transfer via NADH between these positions. Catalytic rates in 1H2O vs. 2H2O revealed a 3-fold difference which was ascribed to a rate-limiting proton addition step. Similarly, a 5-fold decrease in Vmax was found for the reverse reaction when oxoimidazole[2,3-2H2]propionate was compared to unlabeled oxoimidazolepropionate. These data support a mechanism involving water addition across the conjugated double bond system of urocanate, rather than an internal oxidation--reduction process, yet NAD+ is required. A mechanism is proposed which uses electron delocalization in the imidazole nucleus, via an imidazole--NAD adduct, to facilitate water attack and subsequent formation of oxoimidazolepropionate.     

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.     

Transfer of xylose to steroids by rabbit liver microsomes.

Rabbit liver microsomal preparations can transfer xylose from UDP-xylose to estron, 17alpha-estradiol, and 17beta-estradiol, and, in poorer yield, to diethylstilbestrol and p-nitrophenol. No transfer of xylose to estriol, testosterone, epitestosterone or 17alpha-estradiol 3-glucuronide could be demonstrated. The xyloside of [6,7-3H]estrone which was formed by liver microsomes crystallized to constant specific activity with estrone beta-D-xylopyranoside, the chemical preparation of which is described.     

Effect of pressure on deuterium isotope effects of formate dehydrogenase

High pressure causes biphasic effects on the oxidation of formate by yeast formate dehydrogenase as expressed on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by accelerating hydride transfer. The transition state for hydride transfer has a smaller volume than the free formate plus the capturing form of enzyme, with DeltaV(double dagger) = -9.7 +/- 1.0 mL/mol. Pressures above 1.5 kbar decrease capture, reminiscent of effects on the conformational change associated with the binding of nicotinamide adenine dinucleotide (NAD(+)) to yeast alcohol dehydrogenase [Northrop, D. B., and Y. K. Cho (2000) Biochemistry 39, 2406-2412]. The collision complex, E-NAD(+), has a smaller volume than the more tightly bound reactant-state complex, E-NAD(+), with DeltaV = +83.4 +/- 5.2 mL/mol. A comparison of the effects of pressure on the oxidation of normal and deuteroformate shows that the entire isotope effect on hydride transfer, 2.73 +/- 0.20, arises solely from transition-state phenomena, as was also observed previously with yeast alcohol dehydrogense. In contrast, normal primary isotope effects arise solely from different zero-point energies in reactant states, and those that express hydrogen tunneling arise from a mixture of both reactant-state and transition-state phenomena. Moreover, pressure increases the primary intrinsic deuterium isotope effect, the opposite of what was observed with yeast alcohol dehydrogense. The lack of a decrease in the isotope effect is also contrary to empirical precedents from chemical reactions suspected of tunneling and to theoretical constructs of vibrationally enhanced tunneling in enzymatic reactions. Hence, this new experimental design penetrates transition states of enzymatic catalysis as never before, reveals the presence of phenomena foreign to chemical kinetics, and calls for explanations of how enzymes work beyond the tenants of physical organic chemistry.     

15N kinetic isotope effects on uncatalyzed and enzymatic deamination of cytidine.

15N isotope effects and solvent deuterium isotope effects have been measured for the hydrolytic deamination of cytidine catalyzed by Escherichia coli cytidine deaminase and for the uncatalyzed reaction proceeding spontaneously in neutral solution at elevated temperatures. The primary (15)(V/K) arising from the exocyclic amino group for wild-type cytidine deaminase acting on its natural substrate, cytidine, is 1.0109 (in H(2)O, pH 7.3), 1.0123 (in H(2)O, pH 4.2), and 1.0086 (in D(2)O, pD 7.3). Increasing solvent D(2)O content has no substantial effect on k(cat) but enhances k(cat)/K(m), with a proton inventory showing that the fractionation factors of at least two protons increase markedly during the reaction. Mutant cytidine deaminases with reduced catalytic activity show more pronounced (15)N isotope effects of 1.0124 (Glu91Ala), 1.0134 (His102Ala), and 1.0158 (His102Asn) at pH 7.3 in H(2)O, as expected for processes in which the chemical transformation of the substrate becomes more rate determining. The isotope effect of mutant His102Asn is 1.033 after correcting for protonation of the -NH(2) group, and represents the intrinsic isotope effect on C-N bond cleavage. This result allows an estimation of the forward commitment of the reaction with the wild-type enzyme. The observed (15)N kinetic isotope effect of the pyrimidine N-3, for wild-type cytidine deaminase acting on cytidine, is 0.9879, which is consistent with protonation and rehybidization of N-3 with hydroxide ion attack on the adjacent carbon to create a tetrahedral intermediate. These results show that enzymatic deamination of cytidine proceeds stepwise through a tetrahedral intermediate with ammonia elimination as the major rate-determining step. The primary (15)N isotope effects observed for the uncatalyzed reaction at pH 7 (1.0021) and pH 12.5 (1.0034) were found to be insensitive to changing temperatures between 100 and 185 degrees C. These results show that the uncatalyzed and the enzymatic deaminations of cytidine proceed by similar mechanisms, although the commitment to C-N bond breaking is greater for the spontaneous reaction.     

Carbon-13 and deuterium isotope effects on the reaction catalyzed by glyceraldehyde-3-phosphate dehydrogenase

Carbon-13 and deuterium isotope effects have been measured on the reaction catalyzed by rabbit muscle glyceraldehyde-3-phosphate dehydrogenase in an effort to locate the rate-limiting steps. With D-glyceraldehyde 3-phosphate as substrate, hydride transfer is a major, but not the only, slow step prior to release of the first product, and the intrinsic primary deuterium and 13C isotope effects on this step are 5-5.5 and 1.034-1.040, and the sum of the commitments to catalysis is approximately 3. The 13C isotope effects on thiohemiacetal formation and thioester phosphorolysis are 1.005 or less. The intrinsic alpha-secondary deuterium isotope effect at C-4 of the nicotinamide ring of NAD is approximately 1.4; this large normal value (the equilibrium isotope effect is 0.89) shows tight coupling of hydrogen motions in the transition state accompanied by tunneling. With D-glyceraldehyde as substrate, the isotope effects are similar, but the sum of commitments is approximately 1.5, so that hydride transfer is more, but still not solely, rate limiting for this slow substrate. The observed 13C and deuterium equilibrium isotope effects on the overall reaction from the hydrated aldehyde are 0.995 and 1.145, while the 13C equilibrium isotope effect for conversion of a thiohemiacetal to a thioester is 0.994, and that for conversion of a thioester to an acyl phosphate is 0.997. Somewhat uncertain values for the 13C equilibrium isotope effects on aldehyde dehydration and formation of a thiohemiacetal are 1.003 and 1.004.     

Three types of stereospecificity and the kinetic deuterium isotope effect in the oxidative deamination of dopamine as catalyzed by different amine oxidases

When the stereospecifically deuterated dopamine enantiomers, (R)- and (S)-[alpha-2H1]dopamine, are incubated with amine oxidases, the deuterium atom may be either retained to form monodeuterated 3,4-dihydroxyphenylacetaldehyde, or eliminated to produce the nondeuterated or protio-aldehyde product. These two aldehydes can be separated from one another and identified by high-performance liquid chromatography with electrochemical detection. Three types of stereospecific abstraction of a hydrogen from the alpha-carbon of dopamine during deamination have been observed. In the first type, the pro-R hydrogen is removed from the alpha-carbon. Enzymes in this category are mitochondrial monoamine oxidases A and B, as isolated from different tissues and species. The second type of deamination involves the abstraction of pro-S hydrogen from the alpha-carbon of dopamine. Soluble enzymes, such as rat aorta benzylamine oxidase or diamine oxidase from hog kidney and pea seedling, have been found to belong to this group. Bovine plasma amine oxidase exhibits the third type of deamination where no absolute stereospecificity is required. This enzyme catalyzes the oxidation of either (S)- or (R)-[alpha-2H1]dopamine, preferably breaking the C-H bond rather than the C-2H bond in both cases. The kinetic deuterium isotope effect during the deamination of dopamine catalyzed by the different amine oxidases varies greatly; VH/VD ranges from 1.5 to 5.5. The high magnitude of the isotope effect suggests that hydrogen abstraction may be the rate-limiting step (i.e., in reactions catalyzed by benzylamine oxidase and monoamine oxidase). When the isotope effect is low (i.e., for diamine oxidases from hog kidney or pea seedling), it is uncertain if the breaking of the bond is rate limiting.     

Kinetic study of the enzymatic inactivation of progestogens by rat liver microsomes.

The kinetics of enzymatic hydrogenation of synthetic progestogens by female rat liver microsomes with NADPH as hydrogen donor were investigated by means of an optical test. Steroids with high progestational activity (Clauberg test) showed a low hydrogenation rate (Vmax). There also seemed to be a certain correlation between molecular structure and Vmax. The metabolites from incubation with the progestogens were isolated from the micro-preparations, and identified by infrared spectroscopy. It was found that the hydrogenation of the olefinic and carbonylic groups involved the uptake of hydrogen from NADPH.     

Carbon-13 and deuterium isotope effects on the catalytic reactions of biotin carboxylase

13C and 2H kinetic isotope effects have been used to investigate the mechanism of enzymic biotin carboxylation. D(V/K) is 0.50 in 80% D2O at pD 8.0 for the forward reaction and 0.57 at pD 8.5 for the phosphorylation of ADP by carbamoyl phosphate. These values approach the theoretical maximum limit for a reaction in which a proton is transferred from a sulfhydryl to a nitrogen or oxygen base. Therefore, it appears that this portion of the reaction is at or near equilibrium. 13(V/K) at pH 8 is 1.007; the small magnitude of this number suggests that the reaction is almost fully committed by the time the carbon-sensitive steps are reached. There does not appear to be a reverse commitment to the reaction under the conditions in which 13(V/K) was determined. A large forward commitment is consistent with the failure to observe positional isotope exchange from the beta gamma-bridge position to the beta-nonbridge position in [18O4]ATP or washout of 18O from the gamma-nonbridge positions. Transfer of 18O from bicarbonate to inorganic phosphate in the forward reaction was clearly observed, however. These observations suggest that biotin carboxylase exists in two distinct forms which differ in the protonation states of the two active-site bases, one of which is a sulfhydryl. Only when the sulfhydryl is ionized and the second base protonated can catalysis take place. Carboxylation of biotin is postulated to occur via a pathway in which carboxyphosphate in formed by nucleophilic attack of bicarbonate on ATP.(ABSTRACT TRUNCATED AT 250 WORDS)