Steady-state kinetics of substrate binding and iron release in tomato ACC oxidase.

1-Aminocyclopropane-1-carboxylate oxidase (ACC oxidase) catalyzes the last step in the biosynthetic pathway of the plant hormone, ethylene. This unusual reaction results in the oxidative ring cleavage of 1-aminocyclopropane carboxylate (ACC) into ethylene, cyanide, and CO2 and requires ferrous ion, ascorbate, and molecular oxygen for catalysis. A new purification procedure and assay method have been developed for tomato ACC oxidase that result in greatly increased enzymatic activity. This method allowed us to determine the rate of iron release from the enzyme and the effect of the activator, CO2, on this rate. Initial velocity studies support an ordered kinetic mechanism where ACC binds first followed by O2; ascorbate can bind after O2 or possibly before ACC. This kinetic mechanism differs from one recently proposed for the ACC oxidase from avocado.

The catalytic function of bovine lysyl oxidase in the absence of copper.

Bovine lysyl oxidase (BLO) contains two different cofactors, copper (Kagan, H. M. (1986) in Biology of Extracellular Matrix (Mecham, R. P., ed) Vol. 1, pp. 321-398, Academic Press, Orlando, FL) and lysine tyrosyl quinone (LTQ) (Wang, S. X., Mure, M., Medzihradszky, K. F., Burlingame, A. L., Brown, D. E., Dooley, D. M., Smith, A. J., Kagan, H. M., and Klinman, J. P. (1996) Science 273, 1078-1084). By a combination of UV-visible spectroscopy, metal content analysis, and activity measurements, we find that copper-depleted BLO reacts in an irreversible manner with phenylhydrazine, an amine substrate analog, and catalyzes multiple turnovers of the substrate benzylamine. After removal of the majority of enzyme-bound copper, apoBLO exhibits a decrease in the LTQ content, as evidenced by the drop of the 510-520-nm absorbance, suggesting that the copper may play a structural role in stabilizing the LTQ. The remaining intact LTQ in the apoBLO reacted with phenylhydrazine, both in the presence and absence of the chelator, 10 mm 2,2'-dipyridyl. When benzylamine was used as the substrate, the apoBLO turned over at a rate of 50-60% of the native BLO (after correction for the residual copper and the change of LTQ content). Copper contamination from the assay buffer was ruled out by comparison of enzyme activity using different apoBLO concentrations. These studies demonstrate that the mature form of lysyl oxidase retains many of its functions in the absence of copper.

Probes of hydrogen tunneling with horse liver alcohol dehydrogenase at subzero temperatures.

The temperature dependence of steady-state kinetics has been studied with horse liver alcohol dehydrogenase (HLADH) using protonated and deuterated benzyl alcohol as substrates in methanol/water mixtures between +3 and -50 degrees C. Additionally, the competitive isotope effects, k(H)/k(T) and k(D)/k(T), were measured. The studies indicate increasing kinetic complexity for wild-type HLADH at subzero temperatures. Consistent with earlier findings at 25 degrees C [Bahnson et al. (1993) Biochemistry 31, 5503], the F93W mutant shows much less kinetic complexity than the wild-type enzyme between 3 and -35 degrees C. An analysis of noncompetitive deuterium isotope effects and competitive tritium isotope effects leads to the conclusion that the reaction of F93W involves substantial hydrogen tunneling down to -35 degrees C. The effect of methanol on kinetic properties for the F93W mutant was analyzed, showing a dependence of competitive KIEs on the NAD(+) concentration. This indicates a more random bi--bi kinetic mechanism, in comparison to an ordered bi-bi kinetic mechanism in water. Although MeOH also affects the magnitude of the reaction rates and, to some extent, the observed KIEs, the ratio of ln k(H)/k(T) to ln k(D)/k(T) for primary isotope effects has not changed in methanol, and we conclude little or no change in kinetic complexity. Importantly, the degree of tunneling, as shown from the relationship between the secondary k(H)/k(T) and k(D)/k(T) values, is the same in water and MeOH/water mixtures, implicating similar trajectories for H transfer in both solvents. In a recent study of a thermophilic alcohol dehydrogenase [Kohen et al. (1999) Nature 399, 496], it was shown that decreases in temperatures below a transition temperature lead to decreased tunneling. This arises because of a change in protein dynamics below a break point in enzyme activity [Kohen et al. (2000) J. Am. Chem. Soc. 122, 10738-10739]. For the mesophilic HLADH described herein, an opposite trend is observed in which tunneling increases at subzero temperatures. These differences are attributed to inherent differences in tunneling probabilities between 0 and 100 degrees C vs subzero temperatures, as opposed to fundamental differences in protein structure for enzymes from mesophilic vs thermophilic sources. We propose that future investigations of the relationship between protein flexibility and hydrogen tunneling are best approached using enzymes from thermophilic sources.

The role of copper in topa quinone biogenesis and catalysis, as probed by azide inhibition of a copper amine oxidase from yeast.

All known copper amine oxidases (CAOs) contain 2,4,5-trihydroxyphenylalanine quinone (TPQ) as a redox cofactor. TPQ is derived posttranslationally from a specific tyrosine residue within the protein itself, and is utilized by the enzyme to oxidize amines to aldehydes. Several oxidative mechanisms for both turnover and the biogenesis of the cofactor have been proposed in recent years, which differ mainly in the nature of the interaction of oxygen with the enzyme. In this study, azide is used to probe the role of copper in catalysis and biogenesis, especially with respect to potential interactions between the metal and oxygen. During turnover, it is found that azide is a noncompetitive inhibitor with respect to O(2), most consistent mechanistically with oxygen binding off the metal prior to reaction. During biogenesis, it is found that azide likely prohibits ligation of the precursor tyrosine to the copper, thus preventing the formation of this key intermediate. This result is consistent with previous proposals, where the copper-tyrosine unit is the species that undergoes reaction with O(2). In addition, it is found that oxygen consumption is kinetically uncoupled from TPQ formation; this leads to an expanded kinetic model for biogenesis, with important implications for previous results.

Life as aerobes: are there simple rules for activation of dioxygen by enzymes?

Numerous biological systems involve reaction with dioxygen in the absence of readily accessible spectroscopic signals. We have begun to develop a set of "generic" strategies that will allow us to probe the mechanisms of dioxygen activation. In particular, we wish to understand the nature of the dioxygen binding step, the degree to which electron transfer to dioxygen is rate limiting, whether reactive species accumulate during turnover and, finally, whether proton and electron transfer to dioxygen occur as coupled processes. Our strategy will be introduced for an enzyme system that uses only an organic cofactor in dioxygen activation (glucose oxidase). Two key features emerge from studies of glucose oxidase: (1) that formation of the superoxide anion is a major rate-limiting step and (2) that electrostatic stabilization of the superoxide anion plays a key role in catalysis. Similar themes emerge when our protocols are applied to enzymes containing both an active site metal center and an organic cofactor. Finally, enzymes that rely solely on metal centers for substrate functionalization will be discussed. In no instance, thus far, has evidence been found for a direct coupling of proton to electron transfer in the reductive activation of dioxygen.

Mechanisms of biosynthesis of protein-derived redox cofactors.

Prior to 1990, redox cofactors were widely believed to be small molecule, dissociable compounds. In the past 10 years, however, four novel redox cofactors have been discovered, each of which is derived from posttranslational modification of specific amino acids within their cognate enzymes. These include topa quinone, found in copper amine oxidases, lysine tyrosyl quinone, found in lysyl oxidase, tryptophan tryptophylquinone, found in methylamine dehydrogenase, and the cysteine-cross-linked tyrosine found in galactose oxidase. The processes by which these cofactors are formed, called biogenesis, is currently a major focus of mechanistic work in this field. In this review, the latest progress toward elucidating the various biogenesis mechanisms is discussed, along with possible linkages between the chemistries involved in catalysis and biogenesis.

Oxygen kinetic isotope effects in soluble methane monooxygenase.

Soluble methane monooxygenase (sMMO) contains a nonheme, carboxylate-bridged diiron site that activates dioxygen in the catalytic oxidation of hydrocarbon substrates. Oxygen kinetic isotope effects (KIEs) have been determined under steady-state conditions for the sMMO-catalyzed oxidation of CH(3)CN, a liquid substrate analog. Kinetic studies of the steady-state sMMO reaction revealed a competition between fully coupled oxygenase activity, which produced glycolonitrile (HOCH(2)CN) and uncoupled oxidase activity that led to water formation. The oxygen KIE was measured independently for both the oxygenase and oxidase reactions, and values of 1.0152 +/- 0.0007 and 1.0167 +/- 0.0010 were obtained, respectively. The isotope effects and separate dioxygen binding studies do not support irreversible formation of an enzyme-dioxygen Michaelis complex. Additional mechanistic implications are discussed in the context of previous data obtained from single turnover and steady-state kinetic studies.

Crystal structure at 2.5 A resolution of zinc-substituted copper amine oxidase of Hansenula polymorpha expressed in Escherichia coli.

Copper amine oxidases (CAOs) catalyze the two-electron oxidation of primary amines to aldehydes, utilizing molecular oxygen as a terminal electron acceptor. To accomplish this transformation, CAOs utilize two cofactors: a mononuclear copper, and a unique redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ or TOPA quinone). TPQ is derived via posttranslational modification of a specific tyrosine residue within the protein itself. In this study, the structure of an amine oxidase from Hansenula polymorpha has been solved to 2.5 A resolution, in which the precursor tyrosine is unprocessed to TPQ, and the copper site is occupied by zinc. Significantly, the precursor tyrosine directly ligands the metal, thus providing the closest analogue to date of an intermediate in TPQ production. Besides this result, the rearrangement of other active site residues (relative to the mature enzyme) proposed to be involved in the binding of molecular oxygen may shed light on how CAOs efficiently use their active site to carry out both cofactor formation and catalysis.

Reassessment of the active site quino-cofactor proposed to occur in the Aspergillus niger amine oxidase AO-I from the properties of model compounds.

Quino-cofactors have been found in a wide variety of prokaryotic and eukaryotic organisms. Two variants have, thus far, been demonstrated to derive from tyrosine precursors: these are the 2,4, 5-trihydroxyphenylalanine quinone (topa quinone or TPQ) [Janes, S. M. , et al. (1990) Science 248, 98] and an o-quinone analogue containing the side chain of a lysine residue (lysyltyrosine quinone or LTQ) [Wang, S. Z., et al. (1996) Science 273, 1078]. Additionally, a third variant of the family of tyrosine-derived cofactors has been reported to exist in an Aspergillus niger amine oxidase AO-I. This was described as an o-quinone cross-linked to the side chain of a glutamate residue [Frebort, I. (1996) Biochim. Biophys. Acta 1295, 59]. We have synthesized model compounds related to the proposed structure. Characterization of the redox properties for the model compound and spectral properties of its 4-nitrophenylhydrazine derivative lead us to conclude that the cofactor in A. niger amine oxidase AO-I has been misidentified. A TPQ carboxylate ester is considered an unlikely candidate for a biologically functional quino-cofactor.

Investigation of spectroscopic intermediates during copper-binding and TPQ formation in wild-type and active-site mutants of a copper-containing amine oxidase from yeast.

Copper amine oxidases possess the unusual ability to generate autocatalytically their organic cofactor, which is subsequently utilized in turnover. This cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is formed within the active site of these enzymes by the oxidation of a single tyrosine residue. In vitro, copper(II) and oxygen are both necessary and sufficient for the conversion of tyrosine to TPQ. In this study, the biogenesis of TPQ has been characterized in an amine oxidase from Hansenula polymorpha expressed as the apo-enzyme in Escherichia coli. With the WT enzyme, optical absorbances which are copper or oxygen dependent are observed and characterized. Active-site mutants are used to investigate further the nature of these spectral species. Evidence is presented which suggests that tyrosine is activated for reaction with oxygen by liganding to Cu(II). In the following paper in this issue [Schwartz, B., Dove, J. E., and Klinman, J. P. (2000) Biochemistry 39, 3699-3707], the initial reaction of precursor protein with oxygen is characterized kinetically. Taken together, the available data suggest a mechanism for the oxidation of tyrosine to TPQ where the role of the copper is to activate substrate.

Kinetic analysis of oxygen utilization during cofactor biogenesis in a copper-containing amine oxidase from yeast.

A detailed kinetic analysis of oxygen consumption during TPQ biogenesis has been carried out on a yeast copper amine oxidase. O(2) is consumed in a single, exponential phase, the rate of which responds linearly to dissolved oxygen concentration. This behavior is observed up to conditions of maximally obtainable oxygen concentrations. In contrast, no viscosity effect is observed on rate, implicating a high K(m) for O(2). Binding of oxygen appears to occur faster than its consumption and to result in displacement of the precursor tyrosine onto copper to form a charge-transfer species, described in the the preceding paper of this issue [Dove, J. E., Schwartz, B., Williams, N. K., and Klinman, J. P. (2000) Biochemistry 39, 3690-3698). Reaction between this intermediate and O(2) is proposed to occur in a rate-limiting step, and to proceed more rapidly when the tyrosine is deprotonated. This rate-limiting step in cofactor biogenesis does not display a solvent isotope effect and is, thus, uncoupled from proton transfer. Comparisons are drawn between the proposed biogenesis mechanism and that for the oxidation of reduced cofactor during catalytic turnover in the mature enzyme.

Probes of a role for remote binding interactions on hydrogen tunneling in the horse liver alcohol dehydrogenase reaction.

A tunneling contribution to hydride transfer has been demonstrated previously in the oxidation of benzyl alcohol catalyzed by an active-site mutant (F93W) of horse liver alcohol dehydrogenase (LADH) [Bahnson, B. J., et al. (1993) Biochemistry 32, 5503-5507]. Mutation of a residue that lies directly behind the nicotinamide ring of the bound cofactor has further shown that side-chain bulk can contribute to catalytic efficiency and tunneling in a correlated fashion [Bahnson, B. J., et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 12797-12802]. Second site mutations of F93W have now been made at positions more remote from the active site. In particular, we have focused on an isoleucine residue that interacts with the adenine moiety of the NAD(+) cofactor, 20 A from the nicotinamide ring. Replacement of this remote residue with glycine (F93W:I224G), alanine (F93W:I224A), valine (F93W:I224V), and leucine (F93W:I224L) is concluded to destabilize the binding of NAD(+). All double mutants exhibited a K(M) for NAD(+) that is 2-25 times higher than that for the F93W enzyme. However, neither the catalytic efficiency for turnover of benzyl alcohol [k(cat)/K(M(benzyl alcohol))] nor the relationship between the secondary k(H)/k(T) and k(D)/k(T) isotope effects for benzyl alcohol oxidation was significantly affected. The lack of differences observed in the isotope effects indicates that these mutations have little effect on the extent of hydrogen tunneling in the reaction. The complete removal of the side chain at position 224 in the F93W:I224G enzyme resulted in a less than 5% decrease in the ratio of the secondary isotope effects, maintaining the ratio above the semiclassical limit for the indication of tunneling in the reaction. By contrast, K(i) for NAD(+) increased 60-fold for this mutant. The results obtained with F93W:I224G are consistent with remote interactions that affect the association and binding of cofactor in a reactive conformation. However, once this conformation is achieved, hydride transfer and its tunneling component proceed as with the single F93W mutant enzyme, uninfluenced by the remote mutation. Replacement of other side chains, with alpha-carbon positions from about 8 to over 20 A from the C4 position of the nicotinamide ring, demonstrated a similar insensitivity of k(cat)/K(M(benzyl alcohol)) to protein modification. Comparison to earlier studies with active-site mutants of LADH implicates a role for proximal, but not distal, side chains in the modulation of hydrogen tunneling for this enzyme.

Nature of hydrogen transfer in soybean lipoxygenase 1: separation of primary and secondary isotope effects.

Previous measurements of the kinetics of oxidation of linoleic acid by soybean lipoxygenase 1 have indicated very large deuterium isotope effects, but have not been able to distinguish the primary isotope effect from the alpha-secondary effect. To address this question, singly deuterated linoleic acid was prepared, and enantiomerically resolved using the enzyme itself. Noncompetitive measurements of the primary deuterium isotope effect give a value of ca. 40 which is temperature-independent. The enthalpy of activation is low and isotope-independent, and there is a large isotope effect on the Arrhenius prefactor. A very large apparent secondary isotope effect (ca. 2.1) is measured with deuterium in the primary position, but a greatly reduced value (1.1) is observed with protium in the primary position. Mutagenesis of the active site leads to a significant reduction in k(cat) and perturbed isotope effects, in particular, a secondary effect of 5.6 when deuterium is in the primary position. The anomalous secondary isotope effects are shown to arise from imperfect stereoselectivity of hydrogen abstraction which, for the mutant, is attributed to a combination of inverse substrate binding and increased flexibility at the reactive carbon. After correction, a very large primary (76-84) and small secondary (1.1-1.2) kinetic isotope effects are calculated for both mutant and wild-type enzymes. The weight of the evidence is taken to favor hydrogen tunneling as the primary mechanism of hydrogen transfer.

Kinetic parameters for dimeric and tetrameric forms of bovine dopamine beta-monooxygenase and their relationship to non-Michaelis-Menten behavior.

Bovine dopamine beta-monooxygenase has been assayed over a 10,000-fold range in protein concentration, to approximate conditions where the enzyme was shown to be a dimer or tetramer. Michaelis-Menten kinetics are observed with k(cat) and k(cat)/Km for dissociated enzyme reduced 30% and 200-300% relative to tetramer. Addition of chloride ions to very dilute enzyme or the use of intermediate enzyme concentrations causes non-Michaelis-Menten behavior, attributed to an equilibration between dimer and tetramer. This is not expected to contribute to activity within the chromaffin vesicle, where enzyme and chloride ions are at high levels.

An unexpected role for the active site base in cofactor orientation and flexibility in the copper amine oxidase from Hansenula polymorpha.

The role of the active site aspartate base in the aminotransferase mechanism of the copper amine oxidase from the yeast Hansenula polymorpha has been probed by site-directed mutagenesis. The D319E mutant catalyzes the oxidation of methylamine and phenethylamine, but not that of benzylamine. kcat/Km for methylamine is found to be 80-fold reduced compared to that of the wild type. Viscosogen and substrate and solvent deuteration have no effect on this parameter for D319E, which is suggestive of limitation of kcat/Km by a conformational change. This conformational change is proposed to be the movement of the cofactor into a productive orientation upon the binding of substrate. In the absence of substrate, a flipped cofactor orientation is likely, on the basis of resonance Raman evidence that the C5 carbonyl of the cofactor is less solvent accessible than the C3 hydrogen. kcat for D319E methylamine oxidase is reduced 200-fold compared to that of the wild type and is unaffected by substrate deuteration, but displays a substantial solvent isotope effect. A 428 nm absorbance is evident under conditions of saturating methylamine and oxygen with D319E. The D319N mutant is observed to produce a similar absorbance at 430 nm when treated with ammonia despite the fact that this mutant has no amine oxidase activity. Resonance Raman spectroscopy indicates the formation of a covalent ammonia adduct and identifies it as the deprotonated iminoquinone. In contrast, when the D319E mutant is reacted with ammonia, it gives predominantly a 340-350 nm species. This absorbance is ascribed to a localization of the cofactor oxyanion induced by binding of the cation at the active site and not to covalent adduct formation. Resonance Raman spectroscopic examination of the steady state species of D319E methylamine oxidation, in combination with the kinetic data, indicates that the 428 nm species is the deprotonated iminoquinone produced upon reoxidation of the reduced cofactor. A model is proposed in which a central role of the active site base is to position the free cofactor and several enzyme intermediates for optimal activity.

Nature of oxygen activation in glucose oxidase from Aspergillus niger: the importance of electrostatic stabilization in superoxide formation.

Glucose oxidase catalyzes the oxidation of glucose by molecular dioxygen, forming gluconolactone and hydrogen peroxide. A series of probes have been applied to investigate the activation of dioxygen in the oxidative half-reaction, including pH dependence, viscosity effects, 18O isotope effects, and solvent isotope effects on the kinetic parameter Vmax/Km(O2). The pH profile of Vmax/Km(O2) exhibits a pKa of 7.9 +/- 0.1, with the protonated enzyme form more reactive by 2 orders of magnitude. The effect of viscosogen on Vmax/Km(O2) reveals the surprising fact that the faster reaction at low pH (1.6 x 10(6) M-1 s-1) is actually less diffusion-controlled than the slow reaction at high pH (1.4 x 10(4) M-1 s-1); dioxygen reduction is almost fully diffusion-controlled at pH 9.8, while the extent of diffusion control decreases to 88% at pH 9.0 and 32% at pH 5.0, suggesting a transition of the first irreversible step from dioxygen binding at high pH to a later step at low pH. The puzzle is resolved by 18O isotope effects. 18(Vmax/Km) has been determined to be 1.028 +/- 0.002 at pH 5.0 and 1.027 +/- 0.001 at pH 9.0, indicating that a significant O-O bond order decrease accompanies the steps from dioxygen binding up to the first irreversible step at either pH. The results at high pH lead to an unequivocal mechanism; the rate-limiting step in Vmax/Km(O2) for the deprotonated enzyme is the first electron transfer from the reduced flavin to dioxygen, and this step accompanies binding of molecular dioxygen to the active site. In combination with the published structural data, a model is presented in which a protonated active site histidine at low pH accelerates the second-order rate constant for one electron transfer to dioxygen through electrostatic stabilization of the superoxide anion intermediate. Consistent with the proposed mechanisms for both high and low pH, solvent isotope effects indicate that proton transfer steps occur after the rate-limiting step(s). Kinetic simulations show that the model that is presented, although apparently in conflict with previous models for glucose oxidase, is in good agreement with previously published kinetic data for glucose oxidase. A role for electrostatic stabilization of the superoxide anion intermediate, as a general catalytic strategy in dioxygen-utilizing enzymes, is discussed.

Hydrogen tunneling in biology.

The mechanistic details of hydrogen transfer in biological systems are not fully understood. The traditional approach has been to use semiclassical transition-state theory. This theory cannot explain many experimental findings, however, so different approaches that emphasize the importance of quantum mechanics and dynamic effects should also be considered.