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.     

The nature of O2 reactivity leading to topa quinone in the copper amine oxidase from Hansenula polymorpha and its relationship to catalytic turnover

The copper amine oxidases (CAOs) catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain in the active site. Monooxygenation within the ring of tyrosine at a single Cu(II) site is unique in biology and occurs as an early step in the formation of TPQ. The mechanism of this reaction has been further examined in the CAO from Hansenula polymorpha (HPAO). When a Clark electrode fitted to a custom-made, gastight apparatus over a range of initial concentrations of O(2) was used, rates of O(2) consumption at levels greater than air are seen to be reduced relative to earlier results, yielding K(D)(apparent) = 216 microM for O(2). This is consistent with a mechanism in which O(2) binds reversibly to the active site, triggering a conformational change that promotes ligation of tyrosinate to Cu(II). The activated Cu(II)-tyrosinate species has been proposed to react with O(2) in a rate-limiting step, although it was also possible that breakdown of a putative peroxy-intermediate controlled TPQ formation. To test the latter hypothesis, Cu(II)-free HPAO was prepared with 3,5-ring-[(2)H(2)]-tyrosine incorporated throughout the primary sequence. The absence of an isotope effect on the rate of TPQ formation eliminates cleavage of this C-H bond in a proposed Cu(II)-aryl-peroxide intermediate as a rate limiting step. The role of methionine 634, previously found to moderate O(2) binding during the catalytic cycle, is shown here to serve a similar function in TPQ formation. As with catalysis, the rate of TPQ formation correlates with the volume of the hydrophobic side chain at position 634, implicating similar binding sites for O(2) during catalysis and cofactor biogenesis.     

Investigation of Cu(I)-dependent 2,4,5-trihydroxyphenylalanine quinone biogenesis in Hansenula polymorpha amine oxidase

Copper, a mediator of redox chemistries in biology, is often found in enzymes that bind and reduce dioxygen. Among these, the copper amine oxidases catalyze the oxidative deamination of primary amines utilizing a type(II) copper center and 2,4,5-trihydroxyphenylalanine quinone (TPQ), a covalent cofactor derived from the post-translational modification of an active site tyrosine. Previous studies established the dependence of TPQ biogenesis on Cu(II); however, the dependence of cofactor formation on the biologically relevant Cu(I) ion has remained untested. In this study, we demonstrate that the apoform of the Hansenula polymorpha amine oxidase readily binds Cu(I) under anaerobic conditions and produces the quinone cofactor at a rate of 0.28 h(-1) upon subsequent aeration to yield a mature enzyme with kinetic properties identical to the protein product of the Cu(II)-dependent reaction. Because of the change in magnetic properties associated with the oxidation of copper, electron paramagnetic resonance spectroscopy was employed to investigate the nature of the rate-limiting step of Cu(I)-dependent cofactor biogenesis. Upon aeration of the unprocessed enzyme prebound with Cu(I), an axial Cu(II) electron paramagnetic resonance signal was found to appear at a rate equivalent to that for the cofactor. These data provide strong evidence for a rate-limiting release of superoxide from a Cu(II)(O(2)(.)) complex as a prerequisite for the activation of the precursor tyrosine and its transformation for TPQ. As copper is trafficked to intracellular protein targets in the reduced, Cu(I) state, these studies offer possible clues as to the physiological significance of the acquisition of Cu(I) by nascent H. polymorpha amine oxidase.     

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.     

A theoretical study of the dioxygen activation by glucose oxidase and copper amine oxidase

Glucose oxidase (GO) and copper amine oxidase (CAO) catalyze the reduction of molecular oxygen to hydrogen peroxide. If a closed-shell cofactor (like FADH(2) in GO and topaquinone (TPQ) in CAO) is electron donor in dioxygen reduction, the formation of a closed-shell species (H(2)O(2)) is a spin forbidden process. Both in GO and CAO, formation of a superoxide ion that leads to the creation of a radical pair is experimentally suggested to be the rate-limiting step in the dioxygen reduction process. The present density functional theory (DFT) studies suggest that in GO, the creation of the radical pair induces a spin transition by spin orbit coupling (SOC) in O(2)(-)(rad), whereas in CAO, it is induced by exchange interaction with the paramagnetic metal ion (Cu(II)). In the rate-limiting step, this spin-transition is suggested to transform the O(2)(-)(rad)-FADH(2)(+)(rad) radical pair in GO and the Cu(II)-TPQ (triplet) species in CAO, from a triplet (T) to a singlet (S) state. For CAO, a mechanism for the O[bond]O cleavage step in the biogenesis of TPQ is also suggested.     

2,4,5-Trihydroxyphenylalanine quinone biogenesis in the copper amine oxidase from Hansenula polymorpha with the alternate metal nickel

Copper amine oxidase (CAO) is a dual-functioning enzyme that catalyzes the biosynthesis of a self-derived coenzyme and subsequent oxidative deamination of primary amines. The organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), is generated from the post-translational modification of an active site tyrosine (Y405) in a reaction shown to be dependent on both molecular oxygen and a mononuclear copper center. Previous investigations of Cu(II)-dependent cofactor formation in the Hansenula polymorpha amine oxidase (HPAO) provided evidence for the coordination of the precursor tyrosine in forming a ligand-to-metal charge transfer complex as a means of activating the tyrosyl ring for direct attack by triplet-state dioxygen. To further delineate the role of the metal in facilitating this complex series of reactions, apo-HPAO was reconstituted with alternate metals of varying reduction potentials and Lewis acidities [Ni(II), Co(II), Mn(II), Fe(II), and Fe(III)] and the consequence of each substitution on TPQ biogenesis examined. Ni(II) was found to support the transformation of the precursor tyrosine to the quinone cofactor to yield a mature enzyme competent for methylamine oxidation. Detailed kinetic analysis of the mechanism of TPQ biogenesis for the Ni(II)-substituted enzyme has led to the proposal of a direct electron transfer from the metal-coordinated tyrosinate to dioxygen as the dominant rate-limiting step.     

Mechanistic comparison of the cobalt-substituted and wild-type copper amine oxidase from Hansenula polymorpha

A recent report by Mills and Klinman [Mills, S. A., and Klinman, J. P. (2000) J. Am. Chem. Soc. 122, 9897-9904] described the preparation and initial characterization of a cobalt-substituted form of the copper amine oxidase from Hansenula polymorpha (HPAO). This enzyme was found to be fully catalytically active at saturating substrate concentrations, but with a K(m) for O(2) approximately 70-fold higher than that of the copper-containing, wild-type enzyme. Herein, we report a detailed analysis of the mechanism of catalysis for the wild-type and the cobalt-substituted forms of HPAO. Both forms of enzyme are concluded to utilize the same mechanism for oxygen reduction, involving initial, rate-limiting electron transfer from the reduced cofactor of the enzyme to prebound dioxygen. Superoxide formed in this manner is stabilized by the active site metal, facilitating the transfer of a second electron and two protons to form the product hydrogen peroxide. The elevated K(m) for O(2) at the dioxygen binding site in Co-substituted HPAO, relative to that of wild-type HPAO, is proposed to be due to a change in the net charge at the adjacent metal site from +1 (cupric hydroxide) in wild-type enzyme to +2 (cobaltous H(2)O) in cobalt-substituted HPAO.     

The Formation of lysine tyrosylquinone (LTQ) is a self-processing reaction. Expression and characterization of a Drosophila lysyl oxidase

Recent work in our laboratory has established methods for the expression and purification of a recombinant form of Drosophila lysyl oxdidase (rDMLOXL-1) [Molnar, J., Ujfaludi, Z., Fong, S. F. T., Bollinger, J. A., Waro, G., Fogelgren, B., Dooley, D. M., Mink, M., and Csiszar, K. (2005) J. Biol. Chem. 280, 22977-22985]. Previous investigations on the expression and purification of recombinant forms of lysyl oxidase [Kagan, H. M., Reddy, V. B., Panchenko, M. V., Nagan, N., Boak, A. M., Gacheru, S. N., and Thomas, K. (1995) J. Cell. Biochem. 59, 329-338] and lysyl oxidase-like proteins [Jung, S. T., Kim, M. S., Seo, J. Y., Kim, H. C., and Kim, Y. (2003) Protein Expression Purif. 31, 240-246] [Molnar, J., Fong, K. S. K., He, Q. P., Hayashi, K., Kim, Y., Fong, S. F. T., Fogelgren, B., Szauter, K. M., Mink, M., and Csiszar, K. (2003) Biochim. Biophys. Acta 1647, 220-224] have been reported in the literature. However, this is the first time that an expression system has been developed yielding sufficient amounts of a recombinant lysyl oxidase for detailed characterization. rDmLOXL-1 is secreted into the medium from S2 cells, and the protein is readily purified by Cibacon blue affinity chromatography yielding 10 mg of protein per liter of medium. The protein, as initially purified, is inactive and has no detectable copper or cofactor present. Following aerobic dialysis against copper, the protein is active and displays an electronic absorption spectrum with lambda(max) at 504 nm, consistent with the presence of an organic cofactor. Addition of phenylhydrazine to the copper-loaded protein produced a high-affinity adduct with lambda(max) at 454 nm. Comparison of the resonance Raman spectra of this adduct and a phenylhydrazine-labeled model compound of lysine tyrosylquinone (LTQ) establishes that the cofactor in the active, copper-containing enzyme is LTQ. Collectively, the data demonstrate that LTQ biogenesis most likely occurs by self-processing chemistry, requiring only the precursor protein, copper, and oxygen. Electron paramagnetic resonance and circular dichroism spectroscopy were used to characterize the Cu(II) site in rDmLOXL-1. The data are consistent with a tetragonal Cu(II) site with nitrogen and oxygen ligands. Recombinant DmLOXL-1 displayed significant activity toward tropoelastin and a wide variety of amines including polyamines and diamines. beta-aminoproprionitrile (betaAPN), a well-known irreversible inhibitor of mammalian lysyl oxidases, is also a potent inhibitor of rDmLOXL-1. Results from this investigation have important implications for the lysyl oxidase family.     

Kinetic studies of oxygen reactivity in soybean lipoxygenase-1

The reactivity of O(2) with soybean lipoxygenase-1 (SLO) has been examined using a range of kinetic probes. We are able to rule out diffusional encounter of O(2) with protein, an outer-sphere electron transfer to O(2), and proton transfer as rate-limiting steps in k(cat)/K(M)(O(2)) for wild-type enzyme (WT SLO); this restricts the rate-limiting step to either the combination of O(2) with L(*) or a subsequent conformational change. In the Ile(553) --> Phe mutant, which constricts the putative O(2) binding channel [Knapp et al. (2001) J. Am. Chem. Soc. 123, 2931-2932], k(cat)/K(M)(O(2)) decreases by over a factor of 20; yet, this mutant appears to have the same rate-limiting step as WT SLO. It is argued that the slow step on k(cat)/K(M)(O(2)) is the combination of O(2) with L(*), with proximal protein effects determining the rate of reaction. The available data for SLO support the view that enzymes can affect O(2) reactivity without a direct involvement of metal cofactors. The primary role of the Fe(3+) cofactor is to generate an enzyme-bound radical, while the protein is concluded to control the stereo- and regiochemistry of O(2) encounter with this radical.     

Cu(I)-dependent biogenesis of the galactose oxidase redox cofactor

Galactose oxidase is a copper metalloenzyme containing a novel protein-derived redox cofactor in its active site, formed by cross-linking two residues, Cys228 and Tyr272. Previous studies have shown that formation of the tyrosyl-cysteine (Tyr-Cys) cofactor is a self-processing step requiring only copper and dioxygen. We have investigated the biogenesis of cofactor-containing galactose oxidase from pregalactose oxidase lacking the Tyr-Cys cross-link but having a fully processed N-terminal sequence, using both Cu(I) and Cu(II). Mature galactose oxidase forms rapidly following exposure of a pregalactose oxidase-Cu(I) complex to dioxygen (t(1/2) = 3.9s at pH7). In contrast, when Cu(II) is used in place of Cu(I) the maturation process requires several hours (t(1/2) = 5.1 h). EDTA prevents reaction of pregalactose oxidase with Cu(II) but does not interfere with the Cu(I)-dependent biogenesis reaction. The yield of cross-link corresponds to the amount of copper added, although a fraction of the pregalactose oxidase protein is unable to undergo this cross-linking reaction. The latter component, which may have an altered conformation, does not interfere with analysis of cofactor biogenesis at low copper loading. The biogenesis product has been quantitatively characterized, and mechanistic studies have been developed for the Cu(I)-dependent reaction, which forms oxidized, mature galactose oxidase and requires two molecules of O2. Transient kinetics studies of the biogenesis reaction have revealed a pH sensitivity that appears to reflect ionization of a protein group (pKa = 7.3) at intermediate pH resulting in a rate acceleration and protonation of an early oxygenated intermediate at lower pH competing with commitment to cofactor formation. These spectroscopic, kinetic, and biochemical results lead to new insights into the biogenesis mechanism.     

Copper-containing amine oxidases. Biogenesis and catalysis; a structural perspective

This review will focus on how X-ray crystallographic studies of copper-containing amine oxidases have complemented the solution, kinetic, and spectroscopic research on this ubiquitous class of enzymes. These enzymes not only contain a copper ion at the active site, but also a unique organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), which is absolutely required for catalysis. Structural data have not only shed light on the catalytic mechanism of the enzyme, which converts primary amines, using molecular oxygen, to aldehydes, ammonia, and hydrogen peroxide, but also on biogenesis of the cofactor. The cofactor is derived from a tyrosine in the enzyme amino acid sequence and requires only the addition of copper(II) and molecular oxygen in a self-processing event.     

X-ray snapshots of quinone cofactor biogenesis in bacterial copper amine oxidase

The quinone cofactor TPQ in copper amine oxidase is generated by posttranslational modification of an active site tyrosine residue. Using X-ray crystallography, we have probed the copper-dependent autooxidation process of TPQ in the enzyme from Arthrobacter globiformis. Apo enzyme crystals were anaerobically soaked with copper; the structure determined from this crystal provides a view of the initial state: the unmodified tyrosine coordinated to the bound copper. Exposure of the copper-bound crystals to oxygen led to the formation of freeze-trapped intermediates; structural analyses indicate that these intermediates contain dihydroxyphenylalanine quinone and trihydroxyphenylalanine. These are the first visualized intermediates during TPQ biogenesis in copper amine oxidase.     

Kinetic investigations of the rate-limiting step in human 12- and 15-lipoxygenase

Mammalian lipoxygenases have been implicated in several inflammatory disorders; however, the details of the kinetic mechanism are still not well understood. In this paper, human platelet 12-lipoxygenase (12-hLO) and human reticulocyte 15-lipoxygenase-1 (15-hLO) were tested with arachidonic acid (AA) and linoleic acid (LA), respectively, under a variety of changing experimental conditions, such as temperature, dissolved oxygen concentration, and viscosity. The data that are presented show that 12-hLO and 15-hLO have slower rates of product release (k(cat)) than soybean lipoxygenase-1 (sLO-1), but similar or better rates of substrate capture for the fatty acid (k(cat)/K(M)) or molecular oxygen [k(cat)/K(M(O)2)]. The primary, kinetic isotope effect (KIE) for 15-hLO with LA was determined to be temperature-independent and large ((D)k(cat) = 40 +/- 8), over the range of 10-35 degrees C, indicating that C-H bond cleavage is the sole rate-limiting step and proceeds through a tunneling mechanism. The (D)k(cat)/K(M) for 15-hLO, however, was temperature-dependent, consistent with our previous results [Lewis, E. R., Johansen, E., and Holman, T. R. (1999) J. Am. Chem. Soc. 121, 1395-1396], indicating multiple rate-limiting steps. This was confirmed by a temperature-dependent, k(cat)/K(M) solvent isotope effect (SIE), which indicated a hydrogen bond rearrangement step at low temperatures, similar to that of sLO-1 [Glickman, M. H., and Klinman, J. P. (1995) Biochemistry 34, 14077-14092]. The KIE could not be determined for 12-hLO due to its inability to efficiently catalyze LA, but the k(cat)/K(M) SIE was temperature-independent, indicating distinct rate-limiting steps from both 15-hLO and sLO-1.     

Investigation of the catalytic mechanism of the hotdog-fold enzyme superfamily Pseudomonas sp. strain CBS3 4-hydroxybenzoyl-CoA thioesterase

The 4-hydroxybenzoyl-CoA (4-HB-CoA) thioesterase from Pseudomonas sp. strain CBS3 catalyzes the final step of the 4-chlorobenzoate degradation pathway, which is the hydrolysis of 4-HB-CoA to coenzyme A (CoA) and 4-hydroxybenzoate (4-HB). In previous work, X-ray structural analysis of the substrate-bound thioesterase provided evidence of the role of an active site Asp17 in nucleophilic catalysis [Thoden, J. B., Holden, H. M., Zhuang, Z., and Dunaway-Mariano, D. (2002) X-ray crystallographic analyses of inhibitor and substrate complexes of wild-type and mutant 4-hydroxybenzoyl-CoA thioesterase. J. Biol. Chem. 277, 27468-27476]. In the study presented here, kinetic techniques were used to test the catalytic mechanism that was suggested by the X-ray structural data. The time course for the multiple-turnover reaction of 50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase supported a two-step pathway in which the second step is rate-limiting. Steady-state product inhibition studies revealed that binding of CoA (K(is) = 250 ± 70 μM; K(ii) = 900 ± 300 μM) and 4-HB (K(is) = 1.2 ± 0.2 mM) is weak, suggesting that product release is not rate-limiting. A substantial D(2)O solvent kinetic isotope effect (3.8) on the steady-state k(cat) value (18 s(-1)) provided evidence that a chemical step involving proton transfer is the rate-limiting step. Taken together, the kinetic results support a two-chemical pathway. The microscopic rate constants governing the formation and consumption of the putative aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate were determined by simulation-based fitting of a kinetic model to time courses for the substrate binding reaction (5.0 μM 4-HB-CoA and 0.54 μM thioesterase), single-turnover reaction (5 μM [(14)C]-4-HB-CoA catalyzed by 50 μM thioesterase), steady-state reaction (5.2 μM 4-HB-CoA catalyzed by 0.003 μM thioesterase), and transient-state multiple-turnover reaction (50 μM [(14)C]-4-HB-CoA catalyzed by 10 μM thioesterase). Together with the results obtained from solvent (18)O labeling experiments, the findings are interpreted as evidence of the formation of an aspartyl 17-(4-hydroxybenzoyl)anhydride intermediate that undergoes rate-limiting hydrolytic cleavage at the hydroxybenzoyl carbonyl carbon atom.     

Determination of the partial reactions of rotational catalysis in F1-ATPase

Steady-state ATP hydrolysis in the F1-ATPase of the F(O)F1 ATP synthase complex involves rotation of the central gamma subunit relative to the catalytic sites in the alpha3beta3 pseudo-hexamer. To understand the relationship between the catalytic mechanism and gamma subunit rotation, the pre-steady-state kinetics of Mg x ATP hydrolysis in the soluble F1-ATPase upon rapid filling of all three catalytic sites was determined. The experimentally accessible partial reactions leading up to the rate-limiting step and continuing through to the steady-state mode were obtained for the first time. The burst kinetics and steady-state hydrolysis for a range of Mg x ATP concentrations provide adequate constraints for a unique minimal kinetic model that can fit all the data and satisfy extensive sensitivity tests. Significantly, the fits show that the ratio of the rates of ATP hydrolysis and synthesis is close to unity even in the steady-state mode of hydrolysis. Furthermore, the rate of Pi binding in the absence of the membranous F(O) sector is insignificant; thus, productive Pi binding does not occur without the influence of a proton motive force. In addition to the minimal steps of ATP binding, reversible ATP hydrolysis/synthesis, and the release of product Pi and ADP, one additional rate-limiting step is required to fit the burst kinetics. On the basis of the testing of all possible minimal kinetic models, this step must follow hydrolysis and precede Pi release in order to explain burst kinetics. Consistent with the single molecule analysis of Yasuda et al. (Yasuda, R., Noji, H., Yoshida, M., Kinosita, K., and Itoh, H. (2001) Nature 410, 898-904), we propose that the rate-limiting step involves a partial rotation of the gamma subunit; hence, we name this step k(gamma). Moreover, the only model that is consistent with our data and many other observations in the literature suggests that reversible hydrolysis/synthesis can only occur in the active site of the beta(TP) conformer (Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621-628).     

Role of a strictly conserved active site tyrosine in cofactor genesis in the copper amine oxidase from Hansenula polymorpha

The copper amine oxidases catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain within the same active site. The contributions of individual active site amino acids to each of these chemical processes are being delineated. Previously, using the amine oxidase from the yeast Hansenula polymorpha (HPAO), mutations of a strictly conserved and structurally pivotal active site tyrosine (Y305) were studied and their effects on the catalytic cycle demonstrated [Hevel, J. M., Mills, S. A., and Klinman, J. P. (1999) Biochemistry 38, 3683-3693]. This study examines mutations at the same position for their effects on cofactor generation. While the Y305A mutation had moderate effects on the kinetics of catalysis (2.5- and 8-fold effects on k(cat) using ethylamine and benzylamine as substrates), the same mutation slows cofactor formation by approximately 45-fold relative to that of the wild-type (WT). Additionally, the Y305A mutant forms at least two species: primarily TPQ at lower pH and a species with a blue-shifted absorbance at high pH (lambda(max) = 400 nm). The 400 nm species does not react with phenylhydrazine or ethylamine and is stable toward pH buffer exchange, long-term storage (>3 weeks), incubation at high temperatures, or incubation with reductants and colorimetric peroxide quenching reagents. A similar species accumulates appreciably even at approximately neutral pH in the Y305F mutant, despite the fact that the rate of TPQ formation is reduced only 3-fold relative to that of WT HPAO. This small impact of Y305F on the rate of biogenesis contracts with a decrease in k(cat) (using ethylamine as the substrate) of 125-fold. The opposing effects of mutations at position 305 in biogenesis versus catalysis indicate that a single residue can be recruited for different roles during these processes.     

Order of substrate binding in bacterial phenylalanine hydroxylase and its mechanistic implication for pterin-dependent oxygenases

Phenylalanine hydroxylase (PAH) is a pterin-dependent non-heme metalloenzyme that catalyzes the oxidation of phenylalanine to tyrosine, which is the rate-limiting step in the catabolism of Phe. Chromobacterium violaceum phenylalanine hydroxylase (cPAH) has been prepared and its steady-state mechanism has been investigated. The enzyme requires iron for maximal activity. Initial rate measurements, done in the presence of the 6,7-dimethyl-5,6,7,8-tetrahydropterin (DMPH(4)) cofactor, yielded an average apparent k(cat) of 36+/-1 s(-1). The apparent K(M) values measured for the substrates DMPH(4), L-Phe, and O(2) are 44+/-7, 59+/-10, and 76+/-7 microM, respectively. Steady-state kinetic analyses using double-reciprocal plots revealed line patterns consistent with a sequential ter-bi mechanism in which L-Phe is the middle substrate in the order of binding. The occurrence of a line intersection on the double-reciprocal plot abscissa when either pterin or O(2) is saturated suggests that, prior to O(2) binding, DMPH(4) and L-Phe are in associative pre-equilibrium with cPAH. Together with an inhibition study using the oxidized cofactor, 7,8-dimethyl-6,7-dihydropterin, it is conclusive that the mechanism is fully ordered, with DMPH(4) binding the active site first, L-Phe second, and O(2) last. This represents the first conclusive steady-state mechanism for a PAH enzyme.     

Enhancement of In Vivo Tyrosine Hydroxylation in the Rat Adrenal Gland Under Hypoxic Conditions

We examined the effects of hypoxia (8% O2) on in vivo tyrosine hydroxylation, a rate-limiting step for catecholamine synthesis, in the rat adrenal gland. The hydroxylation rate was determined by measuring the rate of accumulation of 3,4-dihydroxyphenylalanine (DOPA) after decarboxylase inhibition. One hour after hypoxic exposure, DOPA accumulation decreased to 60% of control values, but within 2 h it doubled. At 2 h, the apparent Km values for tyrosine and for biopterin cofactor of tyrosine hydroxylase (TH) in the soluble fraction were unchanged, whereas the Vmax value increased by 30%. The content of total or reduced biopterin was unchanged, but the content of tyrosine increased by 80%. Tyrosine administration had little effect on DOPA accumulation under room air conditions but enhanced DOPA accumulation under hypoxia. After denervation of the adrenal gland, the hypoxia-induced increase in DOPA accumulation and in the Vmax value was abolished, whereas the hypoxia-induced increase in tyrosine content was persistent. These results suggest that in vivo tyrosine hydroxylation is enhanced under hypoxia, although availability of oxygen is reduced. The enhancement is the result of both an increase in tyrosine content coupled with increased sensitivity of TH to changes in tyrosine tissue content and of an increase in dependence of TH on tyrosine levels. The increase in the sensitivity of TH and in the Vmax value is neurally induced, whereas the increase in tyrosine content is regulated by a different mechanism.     

18O isotope exchange experiments on phospholipase A2 determined by 13C-NMR: monomeric phosphatidylcholine and micellar phosphatidylethanolamine substrates

Experiments were carried out to determine whether the hydrolytic step or the product release step is the rate-limiting step for non-activated phospholipase A2 hydrolysis (Dennis, E.A. (1983) in The Enzymes, 3rd Edn., Vol 16 (Boyer, P., ed.), pp. 307-353, Academic Press, New York) of mixed micelles of phosphatidylethanolamine and Triton X-100 in the absence of activator phospholipids and of monomeric short chain phosphatidylcholine in the absence of an interface (Lombardo, D. et al. (1986) J. Biol. Chem. 261, 11663-11666). Phospholipase A2-catalyzed exchange of H2(18)O into 1-alkyl-2-[1(13)C]lauroyl-sn-glycero-3-phosphorylethanolamine and into 1-hexanoyl-2-[1-13C]hexanoyl-sn-glycero-3-phosphorylcholine were examined. Incorporation of 18O was detected by the effect of 18O on 13C chemical shifts in 13C-NMR. Both the substrate and products of the reactions were examined for 18O incorporation. 18O was incorporated into the fatty acid product, but no incorporation of 18O into the substrate was found. These results suggest that the hydrolytic step is not followed by a higher energy transition state and that it, or a step before it, is rate-limiting. Coupled with kinetic experiments, this strongly suggests that the hydrolytic step is the rate-limiting step. Thus, the role of micellar and membrane interfaces in phospholipase A2 reactions does not appear to be by aiding product removal from the enzyme active site.