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1.
The crystal structure of the hydroxymandelate synthase (HMS).Co2+.hydroxymandelate (HMA) complex determined to a resolution of 2.3 A reveals an overall fold that consists of two similar beta-barrel domains, one of which contains the characteristic His/His/acid metal-coordination motif (facial triad) found in the majority of Fe2+-dependent oxygenases. The fold of the alpha-carbon backbone closely resembles that of the evolutionarily related enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD) in its closed conformation with a root-mean-square deviation of 1.85 A. HPPD uses the same substrates as HMS but forms instead homogentisate (HG). The active site of HMS is significantly smaller than that observed in HPPD, reflecting the relative changes in shape that occur in the conversion of the common HPP substrate to the respective HMA or HG products. The HMA benzylic hydroxyl and carboxylate oxygens coordinate to the Co2+ ion, and three other potential H-bonding interactions to active site residue side chains are observed. Additionally, it is noted that there is a buried well-ordered water molecule 3.2 A from the distal carboxylate oxygen. The p-hydroxyl group of HMA is within hydrogen-bonding distance of the side chain hydroxyl of a serine residue (Ser201) that is conserved in both HMS and HPPD. This potential hydrogen bond and the known geometry of iron ligation for the substrate allowed us to model 4-hydroxyphenylpyruvate (HPP) in the active sites of both HMS and HPPD. These models suggest that the position of the HPP substrate differs between the two enzymes. In HMS, HPP binds analogously to HMA, while in HPPD, the p-hydroxyl group of HPP acts as a hydrogen-bond donor and acceptor to Ser201 and Asn216, respectively. It is suggested that this difference in the ring orientation of the substrate and the corresponding intermediates influences the site of hydroxylation. 相似文献
2.
Hydroxymandelate synthase (HMS) catalyzes the committed step in the formation of para-hydroxyphenylglycine, a recurrent substructure of polycyclic non-ribosomal peptide antibiotics such as vancomycin. HMS uses the same substrates as 4-hydroxyphenylpyruvate dioxygenase (HPPD), 4-hydroxyphenylpyruvate (HPP) and O2, and also conducts a dioxygenation reaction. The difference between the two lies in the insertion of the second oxygen atom, HMS directing this atom onto the benzylic carbon of the substrate while HPPD hydroxylates the aromatic C1 carbon. We have shown that HMS will bind NTBC, a herbicide/therapeutic whose mode of action is based on the inhibition of HPPD. This occurs despite residue differences at the active site of HMS from those known to contact the inhibitor in HPPD. Moreover, the minimal kinetic mechanism for association of NTBC to HMS differs only slightly from that observed with HPPD. The primary difference is that three charge-transfer species are observed to accumulate during association. The first reversible complex forms with a weak dissociation constant of 520 μM, the subsequent two charge-transfer complexes form with rate constants of 2.7 s−1 and 0.67 s−1. As was the case for HPPD, the final complex has the most intense charge-transfer, is not observed to dissociate, and is unreactive towards dioxygen. 相似文献
3.
(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) catalyzes the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate (HG). This reaction involves decarboxylation, substituent migration, and aromatic oxygenation in a single catalytic cycle. HPPD is a unique member of the alpha-keto acid dependent oxygenases that require Fe(II) and an alpha-keto acid substrate to oxygenate or oxidize an organic molecule. We have examined the reaction coordinate of HPPD from Streptomyces avermitilis using rapid mixing pre-steady-state methods in conjunction with steady-state kinetic analyses. Acid quench reactions and product analysis of homogentisate indicate that HPPD as isolated is fully active and that experiments limited in dioxygen concentration with respect to that of the enzyme do involve a single turnover. These experiments indicate that during the course of one turnover the concentration of homogentisate is stoichiometric with enzyme concentration by approximately 200 ms, well before the completion of the catalytic cycle. Subsequent single turnover reactions were monitored spectrophotometrically under pseudo-first-order and matched concentration reactant conditions. Three spectrophotometrically distinct intermediates are observed to accumulate. The first of these is a relatively strongly absorbing species with maxima at 380 and 480 nm that forms with a rate constant (k(1)) of 7.4 x 10(4) M(-)(1) s(-)(1) and then decays to a second intermediate with a rate constant (k(2)) of 74 s(-)(1). The rate constant for the decay of the second intermediate (k(3)) is 13 s(-)(1) and is concomitant with the formation of the product, homogentisate, based on rapid quench and pre-steady-state fluorescence measurements. The rate constant for this process decreases to 7.6 s(-)(1) when deuterons are substituted for protons in the aromatic ring of the substrate. The release of product from the enzyme is rate limiting and occurs at 1.6 s(-)(1). This final event exhibits a kinetic isotope effect of 2 with deuterium oxide as the solvent, consistent with a solvent isotope effect on V(max) of 2.6 observed in steady-state experiments. 相似文献
4.
Neidig ML Brown CD Kavana M Choroba OW Spencer JB Moran GR Solomon EI 《Journal of inorganic biochemistry》2006,100(12):2108-2116
The alpha-ketoglutate (alpha-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require Fe(II), alpha-KG and dioxygen for catalysis, with the alpha-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the alpha-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an Fe(IV)O intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O(2) to generate this species is the decarboxylation of the alpha-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the alpha-keto acid to Fe(II) and the presence of a 5C Fe(II) site for the O(2) reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate alpha-KG coordination and a 5C Fe(II) site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an approximately 10(5)-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n-->pi( *) transition of the HPPD/Fe(II)/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the alpha-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO(2) is disfavored. 相似文献
5.
Raspail C Graindorge M Moreau Y Crouzy S Lefèbvre B Robin AY Dumas R Matringe M 《The Journal of biological chemistry》2011,286(29):26061-26070
4-Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the conversion of 4-hydroxyphenylpyruvate (HPP) into homogentisate. HPPD is the molecular target of very effective synthetic herbicides. HPPD inhibitors may also be useful in treating life-threatening tyrosinemia type I and are currently in trials for treatment of Parkinson disease. The reaction mechanism of this key enzyme in both plants and animals has not yet been fully elucidated. In this study, using site-directed mutagenesis supported by quantum mechanical/molecular mechanical theoretical calculations, we investigated the role of catalytic residues potentially interacting with the substrate/intermediates. These results highlight the following: (i) the central role of Gln-272, Gln-286, and Gln-358 in HPP binding and the first nucleophilic attack; (ii) the important movement of the aromatic ring of HPP during the reaction, and (iii) the key role played by Asn-261 and Ser-246 in C1 hydroxylation and the final ortho-rearrangement steps (numbering according to the Arabidopsis HPPD crystal structure 1SQD). Furthermore, this study reveals that the last step of the catalytic reaction, the 1,2 shift of the acetate side chain, which was believed to be unique to the HPPD activity, is also catalyzed by a structurally unrelated enzyme. 相似文献
6.
(4-Hydroxyphenyl)pyruvate dioxygenase (HPPD) incorporates both atoms of molecular oxygen into 4-hydroxyphenylpyruvate (HPP) to form homogentisate (HG). This reaction has direct relevance in both medicine and agriculture. In humans, the specific inhibition of HPPD alleviates the symptoms of diseases that arise from tyrosine catabolism defects. However, in plants, the inhibition of HPPD bleaches, stunts, and ultimately kills the organism. The reason for this is that in mammalian metabolism the product HG does not feed into other pathways, whereas in plants it is the precursor for the redox active portion of tocopherols and plastoquinones. There are a number of commercially available herbicides that directly target the inhibition of the HPPD reaction. Plant HPPD however is largely uncharacterized in terms of its catalysis and inhibition reactions. In this study, we examine the catalysis and inhibition of HPPD from Arabidopsis thaliana (AtHPPD). We have expressed AtHPPD and purified the enzyme to high specific activity. This form of HPPD accumulates two transient species in single turnover reactions with the native substrate HPP. These transients appear to be equivalent to intermediates I and III observed in the enzyme from Streptomyces (Johnson-Winters et al. (2005), Biochemistry, 44, 7189-7199). The first intermediate is a relatively strongly absorbing species with maxima at 380 and 490 nm. This species decays to a second intermediate that is fluorescent and has been assigned as the complex of the enzyme with the product, HG. The decay of this intermediate is rate-determining in multiple turnover reactions. The reaction of the enzyme with the analogue of the substrate, phenylpyruvate (PPA), is noncatalytic. A single turnover reaction is observed with this ligand that renders the enzyme oxidized to the ferric form, consumes a stoichiometric amount of dioxygen, and yields 66% phenylacetate as a product. Additional absorbance features at 365 and 670 nm accumulate during inactivation and give the inactivated enzyme a green color but has the same molecular mass as the active enzyme as determined by mass spectrometry. 相似文献
7.
(4-hydroxyphenyl)pyruvate dioxygenase (HPPD) catalyzes the second step in the pathway for the catabolism of tyrosine, the conversion of (4-hydroxyphenyl)pyruvate (HPP) to homogentisate (HG). This reaction involves decarboxylation, substituent migration, and aromatic oxygenation. HPPD is a member of the alpha-keto acid dependent oxygenases that require Fe(II) and an alpha-keto acid substrate to oxygenate an organic molecule. We have examined the binding of ligands to HPPD from Streptomyces avermitilis. Our data show that HPP binds to the apoenzyme and that the apo-HPPD.HPP complex does not bind Fe(II) to generate active holoenzyme. The binding of HPP, phenylpyruvate (PPA), and pyruvate to the holoenzyme produces a weak ligand charge-transfer band at approximately 500 nm that is indicative of bidentate binding of the 1-carboxylate and 2-keto pyruvate oxygen atoms to the active site metal ion. For HPPD from this organism the 4-hydroxyl group of (4-hydroxyphenyl)pyruvate is a requirement for catalysis; no turnover is observed in the presence of phenylpyruvate. The rate constant for the dissociation of Fe(II) from the holoenzyme is 0.0006 s(-)(1) and indicates that this phenomenon is not significantly relevant in steady-state turnover. The addition of HPP and molecular oxygen to the holoenzyme is formally random. The basis of the ordered bi bi steady-state kinetic mechanism previously observed by Rundgren (Rundgren, M. (1977) J. Biol. Chem. 252, 5094-9) is the 3600-fold increase in oxygen reactivity when holo-HPPD is in complex with HPP. This complex reacts with molecular oxygen with a second-order rate constant of 1.4 x 10(5) M(-)(1) s(-)(1) inducing the formation of an intermediate that decays at the catalytically relevant rate of 7.8 s(-)(1). 相似文献
8.
Phenylalanine hydroxylase from Chromobacterium violaceum (CvPheH) is a non-heme iron monooxygenase that catalyzes the hydroxylation of phenylalanine to tyrosine. In this study, we used deuterium kinetic isotope effects to probe the chemical mechanisms of aromatic and benzylic hydroxylation to compare the reactivities of bacterial and eukaryotic aromatic amino acid hydroxylases. The (D) k cat value for the reaction of CvPheH with [(2)H 5]phenylalanine is 1.2 with 6-methyltetrahydropterin and 1.4 with 6,7-dimethyltetrahydropterin. With the mutant enzyme I234D, the (D) k cat value decreases to 0.9 with the latter pterin; this is likely to be the intrinsic effect for addition of oxygen to the amino acid. The isotope effect on the subsequent tautomerization of a dienone intermediate was determined to be 5.1 by measuring the retention of deuterium in tyrosine produced from partially deuterated phenylalanine; this large isotope effect is responsible for the normal effect on k cat. The isotope effect for hydroxylation of the methyl group of 4-CH 3-phenylalanine, obtained from the partitioning of benzylic and aromatic hydroxylation products, is 10. The temperature dependence of this isotope effect establishes the contribution of hydrogen tunneling to benzylic hydroxylation by this enzyme. The results presented here provide evidence that the reactivities of the prokaryotic and eukaryotic hydroxylases are similar and further define the reactivity of the iron center for the family of aromatic amino acid hydroxylases. 相似文献
9.
Leonard M. Hjelmeland Lewis Aronow James R. Trudell 《Biochemical and biophysical research communications》1977,76(2):541-549
Isotope effects for hydroxylation reactions catalyzed by cytochrome P-450 have usually been measured by comparing the overall reaction velocities of deuterated and nondeuterated substrates. Since the rate-limiting step is probably not the single reaction involving covalent bond cleavage, such an approach does not yield information about the primary isotope effect. We measured the primary kinetic isotope effect for benzylic hydroxylation by a method utilizing intramolecular competition, using the symmetrical substrate 1,3-diphenylpropane-1,1-d2. These experiments yield a value of , a larger effect than has previously been reported for benzylic hydroxylations. 相似文献
10.
The regiochemistry and facial stereochemistry of the limonene-6-hydroxylase- (CYP71D18-) mediated hydroxylation of the monoterpene olefin limonene are determined by the absolute configuration of the substrate. (-)-(4S)-Limonene is hydroxylated at the C6 allylic position to give (-)-trans-carveol as the only product, whereas (+)-(4R)-limonene yields multiple hydroxylation products with (+)-cis-carveol predominating. Specifically deuterated limonene enantiomers were prepared to investigate the net stereospecificity of hydroxylation at C6 and the mechanism of multiple product formation. The results of isotopically sensitive branching experiments of competitive and noncompetitive design were consistent with a nondissociative kinetic mechanism, indicating that (4R)-limonene has sufficient freedom of motion within the active site of CYP71D18 to allow formation of either the trans-3- or cis-6-hydroxylated product. However, the kinetic isotope effects resulting from deuterium abstraction were significantly smaller than expected for an allylic hydroxylation, and they did not approach the intrinsic isotope effect. (4S)-Limonene is oxygenated with almost complete stereospecificity for hydrogen abstraction from the trans-6-position, demonstrating rigid orientation during hydrogen abstraction and hydroxyl delivery. The oxygenation of (4R)-limonene leading to the formation of (+/-)-trans-carveol is accompanied by considerable allylic rearrangement and stereochemical scrambling, whereas the formation of (+)-cis-carveol proceeds without allylic rearrangement and with nearly complete stereospecificity for hydrogen abstraction from the cis-6-position. These results demonstrate that a single cytochrome P450 enzyme catalyzes the hydroxylation of small antipodal substrates with distinct stereochemistries and reveal that substrate-dependent positional motion of the intermediate carbon radical (and, therefore, hydroxylation stereospecificity) is determined by active-site binding complementarity. Thus, epimerization and allylic rearrangement are not inherent features of these reactions but occur when loss of active-site complementarity allows increased substrate mobility. 相似文献
11.
The chemical mechanism of hydroxylation, catalyzed by dopamine beta-monooxygenase, has been explored with a combination of secondary kinetic isotope effects and structure-reactivity correlations. Measurement of primary and secondary isotope effects on Vmax/Km under conditions where the intrinsic primary hydrogen isotope effect is known allows calculation of the corresponding intrinsic secondary isotope effect. By this method we have obtained an alpha-deuterium isotope effect, Dk alpha = 1.19 +/- 0.06, with dopamine as substrate. The beta-deuterium isotope effect is indistinguishable from one. The large magnitude of Dk alpha, together with our previous determination of a near maximal primary deuterium isotope effect of 9.4-11, clearly indicates the occurrence of a stepwise process for C-H bond cleavage and C-O bond formation and hence the presence of a substrate-derived intermediate. To probe the nature of this intermediate, a structure-reactivity study was performed by using a series of para-substituted phenylethylamines. Deuterium isotope effects on Vmax and Vmax/Km parameters were determined for all of the substrates, allowing calculation of the rate constants for C-H bond cleavage and product dissociation and dissociation constants for amine and O2 loss from the enzyme-substrate ternary complex. Multiple regression analysis yielded an electronic effect of p = -1.5 for the C-H bond cleavage step, eliminating the possibility of a carbanion intermediate. A negative p value is consistent with formation of either a radical or a carbocation; however, a significantly better correlation is obtained with sigma p rather than sigma p+, implying formation of a radical intermediate via a polarized transition state. Additional effects determined from the regression analyses include steric effects on rate constants for substrate hydroxylation and product release and on KDamine, consistent with a sterically restricted binding site, and a positive electronic effect of p = 1.4 on product dissociation, ascribed to a loss of product from an enzyme-bound Cu(II)-alkoxide complex. These results lead us to propose a mechanism in which O-O homolysis [from a putative Cu(II)-OOH species] and C-H homolysis (from substrate) occur in a concerted fashion, circumventing the formation of a discrete, high energy oxygen species such as hydroxyl radical. The substrate and peroxide-derived radical intermediates thus formed undergo a recombination, kinetically limited by displacement of an intervening water molecule, to give the postulated Cu(II)-alkoxide product complex. 相似文献
12.
Michael L. Neidig Christina D. Brown Michael Kavana Oliver W. Choroba Jonathan B. Spencer Graham R. Moran Edward I. Solomon 《Journal of inorganic biochemistry》2006,100(12):2108
The α-ketoglutate (α-KG)-dependent dioxygenases are a large class of mononuclear non-heme iron enzymes that require FeII, α-KG and dioxygen for catalysis, with the α-KG cosubstrate supplying the two additional electrons required for dioxygen activation. A sub-class of these enzymes exists in which the α-keto acid is covalently attached to the substrate, including (4-hydroxy)mandelate synthase (HmaS) and (4-hydroxyphenyl)pyruvate dioxygenase (HPPD) which utilize the same substrate but exhibit two different general reactivities (H-atom abstraction and electrophilic attack). Previous kinetic studies of Streptomyces avermitilis HPPD have shown that the substrate analog phenylpyruvate (PPA), which only differs from the normal substrate (4-hydroxyphenyl)pyruvate (HPP) by the absence of a para-hydroxyl group on the aromatic ring, does not induce a reaction with dioxygen. While an FeIVO intermediate is proposed to be the reactive species in converting substrate to product, the key step utilizing O2 to generate this species is the decarboxylation of the α-keto acid. It has been generally proposed that the two requirements for decarboxylation are bidentate coordination of the α-keto acid to FeII and the presence of a 5C FeII site for the O2 reaction. Circular dichroism and magnetic circular dichroism studies have been performed and indicate that both enzyme complexes with PPA are similar with bidentate α-KG coordination and a 5C FeII site. However, kinetic studies indicate that while HmaS reacts with PPA in a coupled reaction similar to the reaction with HPP, HPPD reacts with PPA in an uncoupled reaction at an 105-fold decreased rate compared to the reaction with HPP. A key difference is spectroscopically observed in the n → π* transition of the HPPD/FeII/PPA complex which, based upon correlation to density functional theory calculations, is suggested to result from H-bonding between a nearby residue and the carboxylate group of the α-keto acid. Such an interaction would disfavor the decarboxylation reaction by stabilizing electron density on the carboxylate group such that the oxidative cleavage to yield CO2 is disfavored. 相似文献
13.
CYP19A1, or human aromatase catalyzes the conversion of androgens to estrogens in a three-step reaction through the formation of 19-hydroxy and 19-aldehyde intermediates. While the first two steps of hydroxylation are thought to proceed through a high-valent iron-oxo species, controversy exists surrounding the identity of the reaction intermediate that catalyzes the lyase and aromatization reaction. We investigated the kinetic isotope effect on the steady-state turnover of Nanodisc-incorporated human CYP19A1 to explore the mechanisms of this reaction. Our experiments reveal a significant (∼2.5) kinetic solvent isotope effect for the C10–C19 lyase reaction, similar to that of the first two hydroxylation steps (2.7 and 1.2). These data implicate the involvement of Compound 1 as a reactive intermediate in the final aromatization step of CYP19A1. 相似文献
14.
Daiber A Nauser T Takaya N Kudo T Weber P Hultschig C Shoun H Ullrich V 《Journal of inorganic biochemistry》2002,88(3-4):343-352
The mechanism of the heme-thiolate-dependent NADH-NO reductase (P450(NOR)) from Fusarium oxysporum was investigated by kinetic isotope effects including protio, [4S-2H]-, [4R-2H]-, [4,4(2)H(2)]-NADH and stopped-flow measurements. The respective kinetic isotope effects were measured at high NO concentrations and were found to be 1.7, 2.3 and 3.8 indicating a rate-limitation at the reduction step and a moderate stereoselectivity in binding of the cofactor NADH. In a different approach the kinetic isotope effects were determined directly for the reaction of the Fe(III)-NO complex with [4R-2H]- and [4S-2H]-NADH by stopped-flow spectroscopy. The resulting isotope effects were 2.7+/-0.4 for the R-form and 1.1+/-0.1 for the S-form. In addition the 444 nm intermediate could be chemically generated by addition of an ethanolic borohydride solution to the ferric-NO complex at -10 degrees C. In pulse radiolysis experiments a similar absorbing species could be observed when hydroxylamine radicals were generated in the presence of Fe (III) P450(NOR). Based on these results we postulate hydride transfer from NADH to the ferric P450-NO complex resulting in a ferric hydroxylamine-radical or ferryl hydroxylamine-complex and this step, as indicated by the kinetic isotope effects, to be rate-limiting at high concentrations of NO. However, at low concentrations of NO the decay of the 444 nm species becomes the rate-limiting step as envisaged by stopped-flow and optical kinetic measurements in a system in which NO was continuously generated. The last step in the catalytic cycle may proceed by a direct addition of the NO radical to the Fe-hydroxylamine complex or by electron transfer from the NO radical to the ferric-thiyl moiety in analogy to the postulated mechanisms of prostacyclin and thromboxane biosynthesis by the corresponding P450 enzymes. The latter process of electron transfer could then constitute a common step in all heme-thiolate catalyzed reactions. 相似文献
15.
4-Hydroxyphenylpyruvate dioxygenase: a hybrid density functional study of the catalytic reaction mechanism 总被引:2,自引:0,他引:2
Density functional calculations using the B3LYP functional has been used to study the reaction mechanism of 4-hydroxyphenylpyruvate dioxygenase. The first part of the catalytic reaction, dioxygen activation, is found to have the same mechanism as in alpha-ketoglutarate-dependent enzymes; the ternary enzyme-substrate-dioxygen complex is first decarboxylated to the iron(II)-peracid intermediate, followed by heterolytic cleavage of the O-O bond yielding an iron(IV)-oxo species. This highly reactive intermediate attacks the aromatic ring at the C1 position and forms a radical sigma complex, which can either form an arene oxide or undergo a C1-C2 side-chain migration. The arene oxide is found to have no catalytic relevance. The side-chain migration is a two-step process; the carbon-carbon bond cleavage first affords a biradical intermediate, followed by a decay of this species forming the new C-C bond. The ketone intermediate formed by a 1,2 shift of an acetic acid group rearomatizes either at the active site of the enzyme or in solution. The hypothetical oxidation of the aromatic ring at the C2 position was also studied to shed light on the 4-HPPD product specificity. In addition, the benzylic hydroxylation reaction, catalyzed by 4-hydroxymandelate synthase, was also studied. The results are in good agreement with the experimental findings. 相似文献
16.
Formulations of an enzyme mechanism where only a single step is presumed to be isotopically sensitive can be written in terms of forward and reverse commitments to catalysis. These commitments provide a natural and intuitive way of interpreting the observed isotope effects. Unfortunately, when multiple isotopically sensitive steps are present in the mechanism, including effects associated with pre-equilibria of the unbound substrate, the observed V/K kinetic isotope effect is expressed as a complicated expression of the intrinsic rate constants for each step, the interpretation of which is not always immediately obvious. We show here that V/K isotope effects from unbranched or rapid-equilibrium random Michaelis-Menten systems containing multiple isotopically sensitive steps can be written as a weighted average of the intrinsic isotope effects on each step, where this intrinsic isotope effect from each step is the product of the equilibrium isotope effect on the formation of the reacting intermediate for that step and the intrinsic kinetic effect on the forward rate constant for that step, and the weighting factors are simply the reciprocal sum of the forward and reverse commitments for each step i plus unity, 1/(C(fi)+C(ri)+1), equivalent to the sensitivity index [Ray, W.J., 1983. 相似文献
17.
Primary intrinsic deuterium and 13C isotope effects have been determined for liver (LADH) and yeast (YADH) alcohol dehydrogenases with benzyl alcohol as substrate and for yeast aldehyde dehydrogenase (ALDH) with benzaldehyde as substrate. These values have also been determined for LADH as a function of changing nucleotide substrate. As the redox potential of the nucleotide changes from -0.320 V with NAD to -0.258 V with acetylpyridine-NAD, the product of primary and secondary deuterium isotope effects rises from 4 toward 6.5, while the primary 13C isotope effect drops from 1.025 to 1.012, suggesting a trend from a late transition state with NAD to one that is more symmetrical. The values of Dk (again the product of primary and secondary isotope effects) and 13k for YADH with NAD are 7 and 1.023, suggesting for this very slow reaction a more stretched, and thus symmetrical, transition state. With ALDH and NAD, the primary 13C isotope effect on the hydride transfer step lies in the range 1.3-1.6%, and the alpha-secondary deuterium isotope effect on the same step is at least 1.22, but 13C isotope effects on formation of the thiohemiacetal intermediate and on the addition of water to the thio ester intermediate are less than 1%. On the basis of the relatively large 13C isotope effects, we conclude that carbon motion is involved in the hydride transfer steps of dehydrogenase reactions. 相似文献
18.
Anderson MA Cleland WW Huang DT Chan C Shojaei M Christopherson RI 《Biochemistry》2006,45(23):7132-7139
In the pyrimidine biosynthetic pathway, N-carbamyl-L-aspartate (CA-asp) is converted to L-dihydroorotate (DHO) by dihydroorotase (DHOase). The mechanism of this important reaction was probed using primary and secondary 15N and 13C isotope effects on the ring opening of DHO using isotope ratio mass spectrometry (IRMS). The reaction was performed at three different temperatures (25, 37, and 45 degrees C for hamster DHOase; 37, 50, and 60 degrees C for Bacillus caldolyticus), and the product CA-asp was purified for analysis. The primary and secondary kinetic isotope effects for the ring opening of the DHO were determined from analysis of the N and C of the carbamyl group after hydrolysis. In addition, the beta-carboxyl of the residual aspartate was liberated enzymatically by transamination to oxaloacetate with aspartate aminotransferase and then decarboxylation with oxaloacetate decarboxylase. The 13C/12C ratio from the released CO2 was determined by IRMS, yielding a second primary isotope effect. The primary and secondary isotope effects for the reaction catalyzed by DHOase showed little variation between enzymes or temperatures, the primary 13C and 15N isotope effects being approximately 1% on average, while the secondary 13C isotope effect is negligible or very slightly normal (>1.0000). These data indicate that the chemistry is at least partially rate-limiting while the secondary isotope effects suggest that the transition state may have lost some bending and torsional modes leading to a slight lessening of bond stiffness at the carbonyl carbon of the amide of CA-asp. The equilibrium isotope effects for DHO --> CA-asp have also been measured (secondary 13K(eq) = 1.0028 +/- 0.0002, primary 13K(eq) = 1.0053 +/- 0.0003, primary 15K(eq) = 1.0027 +/- 0.0003). Using these equilibrium isotope effects, the kinetic isotope effects for the physiological reaction (CA-asp --> DHO) have been calculated. These values indicate that the carbon of the amide group is more stiffly bonded in DHO while the slightly lesser, but still normal, values of the primary kinetic isotope effect show that the chemistry remains at least partially rate-limiting for the physiological reaction. It appears that the ring opening and closing is the slow step of the reaction. 相似文献
19.
A kinetic model is presented from which steady-state equations are derived that describe the intramolecular competition for the enzymatically mediated hydroxylation of two like groupings of a prochiral substrate. The observed isotope effect in such a system if one of the groupings is isotopically labeled is shown to be a function of three parameters: the equilibrium constant for the catalytically sensitive orientations of the two prochiral groupings at the active site, the intrinsic isotope effect associated with the bond-breaking step, and the relative rates of bond breaking vs. enzyme-substrate dissociation. The expected isotope effects associated with the omega-hydroxylation of racemic, (R)-, and (S)-2-phenylpropane-1,1,1-d3 and the product stereoselectivity associated with the omega-hydroxylation of (R)- and (S)-[1-13C]-2-phenylpropane were determined with microsomal preparations (cytochrome P-450) from untreated and phenobarbital- and beta-naphthoflavone-pretreated male Sprague-Dawley rats. The data from these experiments allow the observed isotope effect to be evaluated in terms of its component parts, i.e., expected isotope effects, product stereoselectivity, and equilibrium constant. These data further suggest that the intramolecular isotope effect is consistent with a hydrogen abstraction recombination mechanism and is largely dependent upon the chemical nature of the porphyrin-Fe-oxene complex but independent of specific apoprotein structure, product stereoselectivity is primarily dependent upon apoprotein structure, and product stereoselectivity is a good measure of the equilibrium constant and both parameters are dependent upon the chirality of the active site. 相似文献
20.
Guengerich FP Miller GP Hanna IH Sato H Martin MV 《The Journal of biological chemistry》2002,277(37):33711-33719
Cytochrome P450 (P450) 2D6 is involved in the oxidation of a large fraction ( approximately 30%) of drugs used by humans and also catalyzes the O-demethylation of the model substrates 3- and 4-methoxyphenethylamine followed by subsequent ring hydroxylation to dopamine. Burst kinetics were not observed; rate-limiting step(s) must occur prior to product formation. Rates of reduction of ferric P450 2D6 were stimulated by 3- or 4-methoxyphenethylamine or the inhibitor quinidine; reduction is not the most rate-limiting step. The non-competitive intramolecular deuterium isotope effect, an estimate of the intrinsic isotope effect, for 4-methoxyphenethylamine O-demethylation was 9.6. Intermolecular non-competitive deuterium isotope effects of 3.1-3.8 were measured for k(cat) and k(cat)/K(m) for both O-demethylation reactions, implicating at least partially rate-limiting C-H bond breaking. Simulation of steady-state kinetic data yielded a catalytic mechanism dominated by the rates of (i) Fe(2+)O(2)(-) protonation (plus O-O bond scission) and (ii) C-H bond breaking, consistent with the appearance of the spectral intermediates in the steady state, attributed to iron-oxygen complexes. However, all the rates of individual steps (or rates of combined steps) are considerably higher than k(cat), and the contributions of several steps must be considered in understanding rates of the P450 2D6 reactions. 相似文献