Ternary Complex Mechanism Research Articles

Characterization of the 2-[(R)-2-hydroxypropylthio]ethanesulfonate dehydrogenase from Xanthobacter strain Py2: product inhibition, pH dependence of kinetic parameters, site-directed mutagenesis, rapid equilibrium inhibition, and chemical modification.

Although the short-chain dehydrogenase/reductase (SDR) superfamily contains a very large number of members defined in annotated databases and by biochemical and structural studies, very few SDR enzymes have been identified that have a homologous partner catalyzing the same reaction but with an opposite stereospecificity. In the present study we have cloned and expressed one of these enzymes, the 2-[(R)-2-hydroxypropylthio]ethanesulfonate (R-HPC) dehydrogenase, that is part of the coenzyme M-dependent pathway of alkene and epoxide metabolism in Xanthobacter strain Py2. Investigation of the kinetic mechanism using product inhibition suggested that a compulsory-ordered ternary complex mechanism was followed. The pH dependence of k(cat)/K(m) indicated the presence of a single ionizable residue of catalytic importance (pK(a) = 6.9) that was proposed to be Y155 of the catalytic triad. Amino acid substitutions of the putative catalytic triad residues produced inactive enzymes (S142C, Y155F, Y155E, and K159A) or enzyme with a greatly decreased activity (S142A). Inhibitors were investigated as probes of the molecular features of R-HPC that contribute to substrate binding. 2-[(S)-2-Hydroxypropylthio]ethanesulfonate (S-HPC) and 2-(2-methyl-2-hydroxypropylthio)ethanesulfonate were found to be competitive inhibitors of R-HPC with K(ic) values close to the K(m) for R-HPC. The arginine-specific modifiers 2,3-butanedione and phenylglyoxal were found to be inactivators, and inactivation could be protected against by the addition of R-HPC. 2,3-Butanedione was found to reduce enzyme activity with R-HPC as a substrate much more dramatically than with substrates that lacked a sulfonate moiety [e.g., 2-propanol, (R)-2-pentanol, and (R)-2-heptanol]. Amino acid analyses of enzyme modified by 2,3-butanedione in the presence and absence of S-HPC suggested protection of a single arginine residue. On the basis of these results, we propose that one or more active site arginines play a key role in substrate binding via an ionic interaction with the sulfonate moiety of R-HPC.

The 11β-hydroxysteroid dehydrogenase type 2 activity in human placental microsomes is inactivated by zinc and the sulfhydryl modifying reagent N-ethylmaleimide

Proper glucocorticoid exposure in utero is vital to normal fetal organ growth and maturation. The human placental 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) catalyzes the unidirectional conversion of cortisol to its inert metabolite cortisone, thereby controlling fetal exposure to maternal cortisol. The present study examined the effect of zinc and the relatively specific sulfhydryl modifying reagent N-ethylmaleimide (NEM) on the activity of 11β-HSD2 in human placental microsomes. Enzyme activity, reflected by the rate of conversion of cortisol to cortisone, was inactivated by NEM (IC 50=10 μM), while the activity was markedly increased by the sulfhydryl protecting reagent dithiothreitol (DTT; EC 50=1 mM). Furthermore, DTT blocked the NEM-induced inhibition of 11β-HSD2 activity. Taken together, these results suggested that the sulfhydryl (SH) group(s) of the microsomal 11β-HSD2 may be critical for enzyme activity. Zn 2+ also inactivated enzyme activity (IC 50=2.5 μM), but through a novel mechanism not involving the SH groups. In addition, prior incubation of human placental microsomes with NAD + (cofactor) but not cortisol (substrate) resulted in a concentration-dependent increase (EC 50=8 μM) in 11β-HSD2 activity, indicating that binding of NAD + to the microsomal 11β-HSD2 facilitated the conversion of cortisol to cortisone. Thus, this finding substantiates the previously proposed concept that a compulsorily ordered ternary complex mechanism may operate for 11β-HSD2, with NAD + binding first, followed by a conformational change allowing cortisol binding with high affinity. Collectively, the present results suggest that cellular mechanisms of SH group modification and intracellular levels of Zn 2+ may play an important role in regulation of placental 11β-HSD2 activity.

Mechanistic studies of methane biogenesis by methyl-coenzyme M reductase: evidence that coenzyme B participates in cleaving the C-S bond of methyl-coenzyme M.

Methyl-coenzyme M reductase (MCR), the key enzyme in methanogenesis, catalyzes methane formation from methyl-coenzyme M (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoBSH). Steady-state and presteady-state kinetics have been used to test two mechanistic models that contrast in the role of CoBSH in the MCR-catalyzed reaction. In class 1 mechanisms, CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage. On the other hand, in class 2 mechanisms, methane is formed in the absence of CoBSH, which functions to regenerate active MCR after methane is released. Steady-state kinetic studies are most consistent with a ternary complex mechanism in which CoBSH binds before methane is formed, as found earlier [Bonacker et al. (1993) Eur. J. Biochem. 217, 587-595]. Presteady-state kinetic experiments at high MCR concentrations are complicated by the presence of tightly bound CoBSH in the purified enzyme. Chemical quench studies in which (14)CH(3)-SCoM is rapidly reacted with active MCRred1 in the presence versus the absence of added CoBSH indicate that CoBSH is required for a single-turnover of methyl-SCoM to methane. Similar single turnover studies using a CoBSH analogue leads to the same conclusion. The results are consistent with class 1 mechanisms in which CoBSH is integrally involved in methane formation and in C-S (methyl-SCoM) bond cleavage and are inconsistent with class 2 mechanisms in which CoBSH binds after methane is formed. These are the first reported pre-steady-state kinetic studies of MCR.

Trehalose-6-phosphate phosphorylase is part of a novel metabolic pathway for trehalose utilization in Lactococcus lactis.

Lactococcus lactis splits phosphorylated trehalose by the action of inorganic phosphate-dependent trehalose-6-phosphate phosphorylase (TrePP) in a novel catabolic pathway. TrePP was found to catalyze the reversible conversion of trehalose 6-phosphate into beta-glucose 1-phosphate and glucose 6-phosphate by measuring intermediate sugar phosphates in cell extracts from trehalose-cultivated lactococci. According to native PAGE and SDS-PAGE, TrePP was shown to be a monomeric enzyme with a molecular mass of 94 kDa. Reaction kinetics suggested that the enzyme follows a ternary complex mechanism with optimal phosphorolysis at 35 degrees C and pH 6.3. The equilibrium constants were found to be 0.026 and 0.032 at pH 6.3 and 7.0, respectively, favoring the formation of trehalose 6-phosphate. The Michaelis-Menten constants of TrePP for trehalose 6-phosphate, inorganic phosphate, beta-glucose 1-phosphate, and glucose 6-phosphate were determined to be 6, 32, 0.9, and 4 mm, respectively. The TrePP-encoding gene, designated trePP, was localized in a putative trehalose operon of L. lactis. This operon includes the gene encoding beta-phosphoglucomutase in addition to three open reading frames believed to encode a transcriptional regulator and two trehalose-specific phosphotransferase system components. The identity of trePP was confirmed by determining the N-terminal amino acid sequence of TrePP and by its overexpression in Escherichia coli and L. lactis, as well as the construction of a lactococcal trePP knockout mutant. Furthermore, both TrePP and beta-phosphoglucomutase activity were detected in Enterococcus faecalis cell extract, indicating that this bacterium exhibits the same trehalose assimilation route as L. lactis.

Copolymerization of methylmethacrylate with styrene using triphenylbismuthoniumylide as radical initiator

AbstractBismuthoniumylide‐initiated radical copolymerization of methylmethacrylate with styrene at 60 ± 0.2°C using dioxane as an inert solvent, follows ideal kinetics (Rp ∝ [ylide]0.5 [MMA]1.0 [sty]1.0), and yields alternating copolymer as evident from NMR spectroscopy. The values of reactivity ratios r1 and r2, calculated from Finemann–Ross method are 0.48 and 0.45, respectively. The system follows ternary molecular complex mechanism. The radical mode of polymerization has been confirmed by ESR spectroscopy and the effect of hydroquinone. The value of activation energy and k/kt are 65.0 KJ mol−1 and 2.5 × 10−5 l mole−1 s−1, respectively. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 80: 2774–2781, 2001

Mechanistic studies on the alkyltransferase activity of serotonin N-acetyltransferase

Background: Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT) catalyzes the first, rate-limiting step in the biosynthesis of the circadian hormone melatonin (5-methoxy- N-acetyltryptamine) from serotonin. Our recent discovery that, in addition to catalyzing the acetyl transfer from acetyl-coenzyme A (acetyl-CoASH) to serotonin, AANAT is also a robust catalyst for the alkyl transfer reaction between CoASH and N-bromoacetyltryptamine has not only opened up a new way to develop cell-permeable AANAT acetyltransferase inhibitors that are valuable in vivo tools in helping elucidate melatonin’s (patho)physiological roles, but has also raised a question – how does AANAT accelerate the alkyl transfer reaction? In this study, mechanistic aspects of the AANAT-catalyzed alkyl transfer reaction were explored by employing CoASH and a series of N-haloacetyltryptamines that were also evaluated for their AANAT acetyltransferase inhibitory activities. Results: Investigation of various N-haloacetyltryptamine analogs showed a similar leaving group effect on the enzymatic and non-enzymatic reaction rates. Steady-state kinetic analyses demonstrated that AANAT alkyltransferase obeys a sequential, ternary complex mechanism, with random substrate binding. Rate versus pH profiles revealed the catalytic importance of an ionizable group with p K a∼7. All those N-haloacetyltryptamines that serve as substrates of AANAT alkyltransferase are also potent (low micromolar) in vitro inhibitors against AANAT acetyltransferase activity. In particular, N-chloroacetyltryptamine was also shown to be a potent inhibitor of intracellular melatonin production in a pineal cell culture assay. Conclusions: This is the first detailed investigation of the alkyltransferase activity associated with an acetyltransferase. Our results indicate that AANAT does not accelerate the alkyl transfer reaction by simple approximation effect as previously proposed for the similar alkyl transfer reaction catalyzed by other acyltransferases. This study has general implications for developing novel inhibitors by taking advantage of the promiscuous alkyltransferase activity associated with several acyltransferases.

Protein phosphatase 1 catalyses the direct hydrolytic cleavage of phosphate monoester in a ternary complex mechanism

The catalytic subunit of the Ser/Thr protein phosphatase 1 (PP1 cat) hydrolyses N-acetyl Arg-Arg-Ala-phosphoThr-Val-Ala ( K M=3.7 mM) in a reaction that is inhibited competitively by inorganic phosphate (P i, K i=1.6 mM) but unaffected by the product peptide alcohol at concentrations up to 3 mM. The enzyme does not catalyse the incorporation of 18O-label from 18O-labelled water into P i whether, or not, the product alcohol is present. The dephosphorylated product alcohol of phosphorylated histone, an alternative substrate for the enzyme, serves as a competitive inhibitor for phosphopeptide hydrolysis ( K i=60 μM) and co-mediates 18O-label exchange into P i in a concentration-dependent manner ( K M=64 μM). These results indicate that hydrolysis occurs through the direct attack of an activated water molecule on the phosphate ester moiety of the substrate in a ternary complex mechanism.

Enzymatic esterification of ethanol and oleic acid — a kinetic study

The formation of ethyl oleate from ethanol and oleic acid using a free or immobilised Rhizomucor miehei lipase as catalyst was evaluated in a biphasic system. Based on a 2(3) factorial design previously developed [1], it was possible to study the reaction kinetics. The data obtained indicated that the reaction follows a Michaelis–Menten kinetics and it is described by the ternary complex mechanism. Based on the proposed model, the kinetic constants to the esterification reaction, without considering substrate inhibition, were determined. The kinetic results showed that the free lipase had the same affinity for both substrates (Km(Et)=1.79 M, Km(Ol)=1.80 M) while the lipase in its immobilised form had higher affinity for oleic acid (Km,app(Et)=1.20 M, Km,app(Ol)=1.16×10−8 M). It was also verified that the specificity was higher in the immobilised lipase system (Ks,app(Et)=2.90 mmol h−1 mg−1 M−1) than in the free one (Ks(Et)=0.637 mmol h−1mg−1 M−1). Diffusional effects were detected for low ethanol and oleic acid concentrations, when using the enzyme in its immobilised form, and related to the effectiveness factor. The integrated Michaelis–Menten equation coupled to the kinetic constants obtained, accord well with experimental results.

P300/CBP-associated Factor Histone Acetyltransferase Processing of a Peptide Substrate: KINETIC ANALYSIS OF THE CATALYTIC MECHANISM

p300/CBP-associated factor (PCAF) is a histone acetyltransferase that plays an important role in the remodeling of chromatin and the regulation of gene expression. It has been shown to catalyze preferentially acetylation of the epsilon-amino group of lysine 14 in histone H3. In this study, the kinetic mechanism of PCAF was evaluated with a 20-amino acid peptide substrate derived from the amino terminus of histone H3 (H3-20) and recombinant bacterially expressed PCAF catalytic domain (PCAF(cat)). The enzymologic behavior of full-length PCAF and PCAF(cat) were shown to be similar. PCAF-catalyzed acetylation of the substrate H3-20 was shown to be specific for Lys-14, analogous to its behavior with the full-length histone H3 protein. Two-substrate kinetic analysis displayed an intersecting line pattern, consistent with a ternary complex mechanism for PCAF. The dead-end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus H3-20. The dead-end analog inhibitor H3-20 K14A was competitive versus H3-20 and uncompetitive versus acetyl-CoA. The potent bisubstrate analog inhibitor H3-CoA-20 was competitive versus acetyl-CoA and noncompetitive versus H3-20. Taken together, these inhibition patterns support an ordered BiBi kinetic mechanism for PCAF in which acetyl-CoA binding precedes H3-20 binding. Viscosity experiments suggest that diffusional release of product is not rate-determining for PCAF catalysis. These results provide a mechanistic framework for understanding the detailed catalytic behavior of an important subset of the histone acetyltransferases and have significant implications for molecular regulation of and inhibitor design for these enzymes.

Kinetic and hydrodynamic studies of the NodL O-acetyl transferase of Rhizobium leguminosarum: a random-order ternary complex mechanism for acetyl transfer by a roughly spherical trimeric protein

The nodL gene product of Rhizobium leguminosarum is required for O-acetylation of diffusible lipo-oligosaccharide signalling factors which are involved in the host-specific nodulation of legume roots. Kinetic studies of the forward reaction, using the substrate analogues chitosan pentaose and chitosan tetraose and the acyl donors acetyl-CoA and propionyl-CoA, and the dead-end inhibitor EtCoA are consistent with a steady-state random-order ternary complex mechanism in which the off rate of the O-acetyl chitosan oligomer appears to be partially rate-determining. Moreover, the linearity of primary double-reciprocal plots favours the view that the interconversion of the ternary complex of NodL and its substrates with that of enzyme and bound products is not significantly faster than k cat. Dissociation constants for coenzyme A and acetyl-CoA were determined by titration microcalorimetry to be 16.5 and 7.2 μM respectively, the latter in agreement with the kinetically derived value of 7.0 μM. The physical state of purified NodL, as determined by equilibrium centrifugation, velocity sedimentation and quasi-elastic light scattering, is that of a roughly spherical, trimeric protein with little tendency to self-associate.

Agonist high and low affinity state ratios predict drug intrinsic activity and a revised Ternary complex mechanism at serotonin 5‐HT2A and 5‐HT2C receptors

Agonist high and low affinity state ratios predict drug intrinsic activity and a revised Ternary complex mechanism at serotonin 5‐HT2A and 5‐HT2C receptors

Catalytic Mechanism of 2-Hydroxybiphenyl 3-Monooxygenase, a Flavoprotein from Pseudomonas azelaica HBP1

2-Hydroxybiphenyl 3-monooxygenase (EC 1.14.13.44) from Pseudomonas azelaica HBP1 is an FAD-dependent aromatic hydroxylase that catalyzes the conversion of 2-hydroxybiphenyl to 2, 3-dihydroxybiphenyl in the presence of NADH and oxygen. The catalytic mechanism of this three-substrate reaction was investigated at 7 degrees C by stopped-flow absorption spectroscopy. Various individual steps associated with catalysis were readily observed at pH 7.5, the optimum pH for enzyme turnover. Anaerobic reduction of the free enzyme by NADH is a biphasic process, most likely reflecting the presence of two distinct enzyme forms. Binding of 2-hydroxybiphenyl stimulated the rate of enzyme reduction by NADH by 2 orders of magnitude. The anaerobic reduction of the enzyme-substrate complex involved the formation of a transient charge-transfer complex between the reduced flavin and NAD(+). A similar transient intermediate was formed when the enzyme was complexed with the substrate analog 2-sec-butylphenol or with the non-substrate effector 2,3-dihydroxybiphenyl. Excess NAD(+) strongly stabilized the charge-transfer complexes but did not give rise to the appearance of any intermediate during the reduction of uncomplexed enzyme. Free reduced 2-hydroxybiphenyl 3-monooxygenase reacted rapidly with oxygen to form oxidized enzyme with no appearance of intermediates during this reaction. In the presence of 2-hydroxybiphenyl, two consecutive spectral intermediates were observed which were assigned to the flavin C(4a)-hydroperoxide and the flavin C(4a)-hydroxide, respectively. No oxygenated flavin intermediates were observed when the enzyme was in complex with 2, 3-dihydroxybiphenyl. Monovalent anions retarded the dehydration of the flavin C(4a)-hydroxide without stabilization of additional intermediates. The kinetic data for 2-hydroxybiphenyl 3-monooxygenase are consistent with a ternary complex mechanism in which the aromatic substrate has strict control in both the reductive and oxidative half-reaction in a way that reactions leading to substrate hydroxylation are favored over those leading to the futile formation of hydrogen peroxide. NAD(+) release from the reduced enzyme-substrate complex is the slowest step in catalysis.

Crystal structure of the histone acetyltransferase domain of the human PCAF transcriptional regulator bound to coenzyme A.

The human p300/CBP-associating factor, PCAF, mediates transcriptional activation through its ability to acetylate nucleosomal histone substrates as well as transcriptional activators such as p53. We have determined the 2.3 A crystal structure of the histone acetyltransferase (HAT) domain of PCAF bound to coenzyme A. The structure reveals a central protein core associated with coenzyme A binding and a pronounced cleft that sits over the protein core and is flanked on opposite sides by the N- and C-terminal protein segments. A correlation of the structure with the extensive mutagenesis data for PCAF and the homologous yeast GCN5 protein implicates the cleft and the N- and C-terminal protein segments as playing an important role in histone substrate binding, and a glutamate residue in the protein core as playing an essential catalytic role. A structural comparison with the coenzyme-bound forms of the related N-acetyltransferases, HAT1 (yeast histone acetyltransferase 1) and SmAAT (Serratia marcescens aminoglycoside 3-N-acetyltransferase), suggests the mode of substrate binding and catalysis by these enzymes and establishes a paradigm for understanding the structure-function relationships of other enzymes that acetylate histones and transcriptional regulators to promote activated transcription.

Kinetics of transhydrogenase reaction catalyzed by the mitochondrial NADH-ubiquinone oxidoreductase (Complex I) imply more than one catalytic nucleotide-binding sites

The steady-state kinetics of the transhydrogenase reaction (the reduction of acetylpyridine adenine dinucleotide (APAD +) by NADH, DD transhydrogenase) catalyzed by bovine heart submitochondrial particles (SMP), purified Complex I, and by the soluble three-subunit NADH dehydrogenase (FP) were studied to assess a number of the Complex I-associated nucleotide-binding sites. Under the conditions where the proton-pumping transhydrogenase (EC 1.6.1.1) was not operating, the DD transhydrogenase activities of SMP and Complex I exhibited complex kinetic pattern: the double reciprocal plots of the velocities were not linear when the substrate concentrations were varied in a wide range. No binary complex (ping-pong) mechanism (as expected for a single substrate-binding site enzyme) was operating within any range of the variable substrates. ADP-ribose, a competitive inhibitor of NADH oxidase, was shown to compete more effectively with NADH ( K i=40 μM) than with APAD + ( K i=150 μM) in the transhydrogenase reaction. FMN redox cycling-dependent, FP catalyzed DD transhydrogenase reaction was shown to proceed through a ternary complex mechanism. The results suggest that Complex I and the simplest catalytically competent fragment derived therefrom (FP) possess more than one nucleotide-binding sites operating in the transhydrogenase reaction.

Molecular Basis for the Stabilization and Inhibition of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by t-Butanol

The steady-state cleavage of catechols by 2,3-dihydroxybiphenyl 1, 2-dioxygenase (DHBD), the extradiol dioxygenase of the biphenyl biodegradation pathway, was investigated using a highly active, anaerobically purified preparation of enzyme. The kinetic data obtained using 2,3-dihydroxybiphenyl (DHB) fit a compulsory order ternary complex mechanism in which substrate inhibition occurs. The Km for dioxygen was 1280 +/- 70 microM, which is at least 2 orders of magnitude higher than that reported for catechol 2,3-dioxygenases. Km and Kd for DHB were 22 +/- 2 and 8 +/- 1 microM, respectively. DHBD was subject to reversible substrate inhibition and mechanism-based inactivation. In air-saturated buffer, the partition ratios of catecholic substrates substituted at C-3 were inversely related to their apparent specificity constants. Small organic molecules that stabilized DHBD most effectively also inhibited the cleavage reaction most strongly. The steady-state kinetic data and crystallographic results suggest that the stabilization and inhibition are due to specific interactions between the organic molecule and the active site of the enzyme. t-Butanol stabilized the enzyme and inhibited the cleavage of DHB in a mixed fashion, consistent with the distinct binding sites occupied by t-butanol in the crystal structures of the substrate-free form of the enzyme and the enzyme-DHB complex. In contrast, crystal structures of complexes with catechol and 3-methylcatechol revealed relationships between the binding of these smaller substrates and t-butanol that are consistent with the observed competitive inhibition.

Purification and characterization of a flavoprotein involved in the degradation of epoxyalkanes by Xanthobacter Py2.

Recently a newly discovered pyridine nucleotide-disulfide oxidoreductase was reported to be essential for the degradation of epoxyalkanes by the Xanthobacter Py2 [Swaving, J., De Bont, J. A. M., Westphal, A. & De Kok, A. (1996) J. Bacteriol. 178, 6644-6646]. The disulfide oxidoreductase has now been purified from propene-grown Xanthobacter Py2. This enzyme (component II) is a NADPH-dependent FAD-containing homodimeric protein. The physiological substrate for this enzyme is unknown. The enzyme was active with the following dithiol substrates in decreasing order: 1,3-propanedithiol, reduced lipoamide and dithiothreitol, and inactive with glutathione and monothiols. In the reversed direction, only activity with 5,5'-dithiobis(2-nitrobenzoate) could be measured. Compared with other disulfide reductases it has a high activity with 5,5'-dithiobis(2-nitrobenzoate) and a low diaphorase and oxidase activity. Steady-state kinetic studies at pH 8.5 with 1,3-propanedithiol show that the enzyme operates by a ternary complex mechanism in the direction of NADP+ reduction. Anaerobic incubation of the enzyme with 1,3-propanedithiol resulted in slow reduction of the enzyme to yield the thiolate-FAD charge-transfer complex, the rate depending on the pH. At pH 7, where reduction was not detectable within 2 h, rapid mixing of NADP+ with the enzyme-propanedithiol mixture resulted in the formation of a complex between the reduced enzyme and NADP+ within the dead time of the instrument (5.6 ms). This is followed by slow formation of NADPH, concomitant with the appearance of the flavin C(4a)-thiol adduct, as judged from the spectral changes. This suggests that the rate-limiting step is the transfer of a hydride ion from the half-reduced enzyme to NADP+. Stopped-flow experiments involving reduction by NADPH show a biphasic behavior. The rapid formation (k(obs) = 40 s(-1)) of a transient intermediate with little absorption decrease at 460 nm and long wavelength absorption was followed by the slow formation (k(obs) = 4 s(-1)) of a species characterized as the thiolate-FAD charge-transfer complex with bound NADP+. Some formation of the FAD C(4a)-thiol adduct was also observed. Photoreduction in the presence of deazaflavin results in rapid bleaching at 450 nm, followed by the slow formation of a stable semiquinone. Full reduction could not be achieved, either by photoreduction or with NADPH, and was incomplete even with dithionite or NADPH in the presence of arsenite. The results indicate a low redox potential of the FAD and a slow rate of electron transfer from the pyridine nucleotide to the redox active disulfide and vice versa. From a sequence alignment with other disulfide reductases, it appears that the active site His-Glu diad is absent in this enzyme. The kinetic and spectral features described above will be discussed in this context.

Kinetic Analysis of the Catalytic Mechanism of Serotonin N-Acetyltransferase (EC 2.3.1.87)

Serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AANAT, EC 2.3.1.87) is the penultimate enzyme in melatonin biosynthesis. This enzyme is of special biological interest because large changes in its activity drive the large night/day rhythm in circulating melatonin in vertebrates. In this study the kinetic mechanism of AANAT action was studied using bacterially expressed glutathione S-transferase (GST)-AANAT fusion protein. The enzymologic behavior of GST-AANAT and cleaved AANAT was essentially identical. Two-substrate kinetic analysis generated an intersecting line pattern characteristic of a ternary complex mechanism. The dead end inhibitor analog desulfo-CoA was competitive versus acetyl-CoA and noncompetitive versus tryptamine. Tryptophol was not an alternative substrate but was a dead end competitive inhibitor versus tryptamine and an uncompetitive inhibitor versus acetyl-CoA, indicative of an ordered binding mechanism requiring binding of acetyl-CoA first. N-Acetyltryptamine, a reaction product, was a noncompetitive inhibitor versus tryptamine and uncompetitive with respect to acetyl-CoA. Taken together these results support an ordered BiBi ternary complex (sequential) kinetic mechanism for AANAT and provide a framework for inhibitor design.

NAD(P)H-dependent aldose reductase from the xylose-assimilating yeast Candida tenuis. Isolation, characterization and biochemical properties of the enzyme.

During growth on d-xylose the yeast Candida tenuis produces one aldose reductase that is active with both NADPH and NADH as coenzyme. This enzyme has been isolated by dye ligand and anion-exchange chromatography in yields of 76%. Aldose reductase consists ofa single 43 kDa polypeptide with an isoelectric point of 4.70. Initial velocity, product inhibition and binding studies are consistent with a compulsory-ordered, ternary-complex mechanism with coenzyme binding first and leaving last. The catalytic efficiency (kcat/Km) in d-xylose reduction at pH 7 is more than 60-fold higher than that in xylitol oxidation and reflects significant differences in the corresponding catalytic centre activities as well as apparent substrate-binding constants. The enzyme prefers NADP(H) approx. 2-fold to NAD(H), which is largely due to better apparent binding of the phosphorylated form of the coenzyme. NADP+ is a potent competitive inhibitor of the NADH-linked aldehyde reduction (Ki 1.5 microM), whereas NAD+ is not. Unlike mammalian aldose reductase, the enzyme from C. tenuis is not subject to oxidation-induced activation. Evidence of an essential lysine residue located in or near the coenzyme binding site has been obtained from chemical modification of aldose reductase with pyridoxal 5'-phosphate. The results are discussed in the context of a comparison of the enzymic properties of yeast and mammalian aldose reductase.

Catalytic mechanism of the oxidative demethylation of 4-(methoxymethyl)phenol by vanillyl-alcohol oxidase. Evidence for formation of a p-quinone methide intermediate.

The catalytic mechanism for the oxidative demethylation of 4-(methoxymethyl)phenol by the covalent flavoprotein vanillyl-alcohol oxidase was studied. Using H218O, it was found that the carbonylic oxygen atom from the product 4-hydroxybenzaldehyde originates from a water molecule. Oxidation of vanillyl alcohol did not result in any incorporation of 18O. Enzyme-monitored turnover experiments revealed that for both substrates a process involving flavin reduction is rate determining. During anaerobic reduction of vanillyl-alcohol oxidase by 4-(methoxymethyl)phenol, a relatively stable spectral intermediate is formed. Deconvolution of its spectral characteristics showed a typical pH-independent absorption maximum at 364 nm (epsilon364 nm = 46 mM-1 cm-1). A similar transient species was observed upon anaerobic reduction by vanillyl alcohol. The rate of flavin reduction and synchronous intermediate formation by 4-(methoxymethyl)phenol is 3.3 s-1 and is fast enough to account for turnover (3.1 s-1). The anaerobic decay of the intermediate was too slow (0.01 s-1) to be of catalytical relevance. The reduced binary complex is rapidly reoxidized (1.5 x 10(5) M-1 s-1) and is accompanied with formation and release of product. Oxidation of free-reduced enzyme is an even faster process (3.1 x 10(5) M-1 s-1). The kinetic data for the oxidative demethylation of 4-(methoxymethyl)phenol are in accordance with a ternary complex mechanism in which the reduction rate is rate-limiting. It is proposed that, upon reduction, a binary complex is produced composed of the p-quinone methide of 4-(methoxymethyl)phenol and reduced enzyme.

The reduction of acetylpyridine adenine dinucleotide by NADH: is it a significant reaction of proton-translocating transhydrogenase, or an artefact?

Transhydrogenase is a proton pump. It has separate binding sites for NAD +/NADH (on domain I of the protein) and for NADP +/NADPH (on domain III). Purified, detergent-dispersed transhydrogenase from Escherichia coli catalyses the reduction of the NAD + analogue, acetylpyridine adenine dinucleotide (AcPdAD +), by NADH at a slow rate in the absence of added NADP + or NADPH. Although it is slow, this reaction is surprizing, since transhydrogenase is generally thought to catalyse hydride transfer between NAD(H) – or its analogues and NADP(H) – or its analogues, by a ternary complex mechanism. It is shown that hydride transfer occurs between the 4 A position on the nicotinamide ring of NADH and the 4 A position of AcPdAD +. On the basis of the known stereospecificity of the enzyme, this eliminates the possibilities of transhydrogenation (a) from NADH in domain I to AcPdAD + wrongly located in domain III; and (b) from NADH wrongly located in domain III to AcPdAD + in domain I. In the presence of low concentrations of added NADP + or NADPH, detergent-dispersed E. coli transhydrogenase catalyses the very rapid reduction of AcPdAD + by NADH. This reaction is cyclic; it takes place via the alternate oxidation of NADPH by AcPdAD + and the reduction of NADP + by NADH, while the NADPH and NADP + remain tightly bound to the enzyme. In the present work, it is shown that the rate of the cyclic reaction and the rate of reduction of AcPdAD + by NADH in the absence of added NADP +/NADPH, have similar dependences on pH and on MgSO 4 concentration and that they have a similar kinetic character. It is therefore suggested that the reduction of AcPdAD + by NADH is actually a cyclic reaction operating, either with tightly bound NADP +/NADPH on a small fraction (<5%) of the enzyme, or with NAD +/NADH (or AcPdAD +/AcPdADH) unnaturally occluded within the domain III site. Transhydrogenase associated with membrane vesicles (chromatophores) of Rhodospirillum rubrum also catalyses the reduction of AcPdAD + by NADH in the absence of added NADP +/NADPH. When the chromatophores were stripped of transhydrogenase domain I, that reaction was lost in parallel with `normal reverse' transhydrogenation (e.g., the reduction of AcPdAD + by NADPH). The two reactions were fully recovered upon reconstitution with recombinant domain I protein. However, after repeated washing of the domain I-depleted chromatophores, reverse transhydrogenation activity (when assayed in the presence of domain I) was retained, whereas the reduction of AcPdAD + by NADH declined in activity. Addition of low concentrations of NADP + or NADPH always supported the same high rate of the NADH→AcPdAD + reaction independently of how often the membranes were washed. It is concluded that, as with the purified E. coli enzyme, the reduction of AcPdAD + by NADH in chromatophores is a cyclic reaction involving nucleotides that are tightly bound in the domain III site of transhydrogenase. However, in the case of R. rubrum membranes it can be shown with some certainty that the bound nucleotides are NADP + or NADPH. The data are thus adequately explained without recourse to suggestions of multiple nucleotide-binding sites on transhydrogenase.