Hydrogen tunneling in enzyme reactions.

Primary and secondary protium-to-tritium (H/T) and deuterium-to-tritium (D/T) kinetic isotope effects for the catalytic oxidation of benzyl alcohol to benzaldehyde by yeast alcohol dehydrogenase (YADH) at 25 degrees Celsius have been determined. Previous studies showed that this reaction is nearly or fully rate limited by the hydrogen-transfer step. Semiclassical mass considerations that do not include tunneling effects would predict that kH/kT = (kD/kT)3.26, where kH, kD, and kT are the rate constants for the reaction of protium, deuterium, and tritium derivatives, respectively. Significant deviations from this relation have now been observed for both primary and especially secondary effects, such that experimental H/T ratios are much greater than those calculated from the above expression. These deviations also hold in the temperature range from 0 to 40 degrees Celsius. Such deviations were previously predicted to result from a reaction coordinate containing a significant contribution from hydrogen tunneling.

A new redox cofactor in eukaryotic enzymes: 6-hydroxydopa at the active site of bovine serum amine oxidase.

An active site, cofactor-containing peptide has been obtained in high yield from bovine serum amine oxidase. Sequencing of this pentapeptide indicates: Leu-Asn-X-Asp-Tyr. Analysis of the peptide by mass spectrometry, ultraviolet-visible spectroscopy, and proton nuclear magnetic resonance leads to the identification of X as 6-hydroxydopa. This result indicates that, contrary to previous proposals, pyrroloquinoline quinone is not the active site cofactor in mammalian copper amine oxidases. Although 6-hydroxydopa has been implicated in neurotoxicity, the data presented suggest that this compound has a functional role at an enzyme active site.

A crosslinked cofactor in lysyl oxidase: redox function for amino acid side chains.

A previously unknown redox cofactor has been identified in the active site of lysyl oxidase from the bovine aorta. Edman sequencing, mass spectrometry, ultraviolet-visible spectra, and resonance Raman studies showed that this cofactor is a quinone. Its structure is derived from the crosslinking of the epsilon-amino group of a peptidyl lysine with the modified side chain of a tyrosyl residue, and it has been designated lysine tyrosylquinone. This quinone appears to be the only example of a mammalian cofactor formed from the crosslinking of two amino acid side chains. This discovery expands the range of known quino-cofactor structures and has implications for the mechanism of their biogenesis.

Quantum mechanical effects in enzyme-catalysed hydrogen transfer reactions.

Hydrogen tunneling at room temperature has recently been demonstrated in the reactions catalysed by yeast alcohol dehydrogenase and bovine serum amine oxidase. These results suggest that quantum mechanical effects may be a fundamental and pervasive feature of enzyme-catalysed hydrogen transfer reactions. Our ability to detect such behavior introduces a new probe for the investigation of reaction barrier shape and protein dynamics in enzyme catalysis.

Copper amine oxidase from Hansenula polymorpha: the crystal structure determined at 2.4 A resolution reveals the active conformation.

BACKGROUND: Copper-containing amine oxidases (CAOs) are widespread in nature. These enzymes oxidize primary amine substrates to the aldehyde product, reducing molecular oxygen to hydrogen peroxide in the process. CAOs contain one type 2 copper atom and topaquinone (TPQ), a modified tyrosine sidechain utilized as a redox cofactor. The methylamine oxidase from the yeast Hansenula polymorpha (HPAO) is an isoform of CAO with a preference for small aliphatic amine or phenethylamine substrates. The enzyme is dimeric with a subunit molecular weight of 78 kDa. Structural studies are directed at understanding the basis for cofactor biogenesis and catalytic efficiency. RESULTS: The X-ray crystal structure of HPAO has been solved at 2.4 A resolution by a combination of molecular replacement and single isomorphous replacement followed by refinement using sixfold symmetry averaging. The electron density at the catalytic site shows that the TPQ conformation corresponds to that of the active form of the enzyme. Two channels, one on either side of TPQ, are observed in the structure that provide access between the active site and the bulk solvent. CONCLUSIONS: The structure shows TPQ in a position poised for catalysis. This is the first active CAO structure to reveal this conformation and may help further our understanding of the catalytic mechanism. On the substrate side of TPQ a water-containing channel leading to the protein surface can serve as an entrance or exit for substrate and product. On the opposite side of TPQ there is direct access from the bulk solvent of the dimer interface by which molecular oxygen may enter and hydrogen peroxide depart. In addition, a network of conserved water molecules has been identified which may function in the catalytic mechanism.

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.

Limited proteolysis of Hansenula polymorpha yeast amine oxidase: isolation of a C-terminal fragment containing both a copper and quino-cofactor.

Limited proteolysis of recombinant Hansenula polymorpha yeast amino oxidase produces a 48 kDa fragment which corresponds to the C-terminal two-thirds of the protein. The fragment contains both TOPA (2,4,5-trihydroxyphenylalanine) and copper, as well as the histidine ligands implicated in copper binding. The fragment is proposed to be the domain responsible for cofactor production in yeast amine oxidase.

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.

Reductive trapping of substrate to methylamine oxidase from Arthrobacter P1.

Methylamine oxidase (EC 1.4.3.6) from Arthrobacter P1 was inactivated by NaCNBH3 in the presence of [14C]benzylamine, leading to the incorporation of 1 mol of radiolabeled substrate/mol of enzyme subunit at complete inactivation. By contrast, no labeling of enzyme was observed using [3H]NaCNBH3 as reductant. These results are analogous to those previously reported for the eukaryotic enzyme, bovine serum plasma amine oxidase [(1987) J. Biol. Chem. 262, 962-965]. The observed pattern of labeling is consistent with the presence of dicarbonyl cofactor at the active site of methylamine oxidase. Further, these studies suggest that our reductive trapping technique, in which the pattern of radiolabeling of an enzyme is compared using C-14 substrate vs tritiated reductant, may serve as a general assay for covalently bound dicarbonyl structures.

Status of the cofactor identity in copper oxidative enzymes.

Much conflicting data have appeared in the literature regarding the nature of the active site structures responsible for catalysis in three classes of copper enzymes: the copper amine oxidases, dopamine beta-monooxygenase and galactose oxidase. Although pyrroloquinoline quinone has been proposed to be the active site cofactor in each instance, new findings indicate this is not the case. Instead, recently available data indicate a spectrum of strategies for substrate activation, which range from direct metal catalysis (dopamine beta-monooxygenase) to the involvement of protein-derived radicals (galactose oxidase) and protein-derived quinones (copper amine oxidases).

Identification of the quinone cofactor in a lysyl oxidase from Pichia pastoris.

A copper amine oxidase from Pichia pastoris is the only known non-mammalian lysyl oxidase [Tur, S.S. and Lerch, K. (1988) FEBS Lett. 238, 74-76]. Recently, the cofactor in mammalian lysyl oxidase has been identified as a novel lysine tyrosylquinone moiety [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]. In order to identify the cofactor in P. pastoris lysyl oxidase, we have isolated the phenylhydrazone-derivative of the active-site peptide. This peptide has the active-site sequence conserved among topa quinone containing amine oxidases. The resonance Raman spectra of the phenylhydrazone derivatives of the enzyme, active-site peptide, and a topa quinone model compound are essentially identical. Collectively, these results establish that P. pastoris lysyl oxidase is a topa quinone enzyme.

Pyrroloquinoline quinone: a new redox cofactor in eukaryotic enzymes.

During the past decade pyrroloquinoline quinone has been shown to be a new redox cofactor for a range of bacterial alcohol dehydrogenases. Recent studies suggest that this cofactor may also be covalently bound to the active site of the eukaryotic copper amine oxidases. In this mini-review we present the evidence in support of pyrroloquinoline quinone as a novel eukaryotic cofactor. As a result of mechanistic advances during the last three years, together with a re-examination of previously existing data, a working model for the role of pyrroloquinoline quinone in enzyme-catalyzed amine oxidation reactions can be proposed.

Acid-base catalysis in the yeast alcohol dehydrogenase reaction.

The effect of pH on steady state kinetic parameters for the yeast alcohol dehydrogenase-catalyzed reduction of aldehydes and oxidation of alcohols has been studied. The oxidation of p-CH3 benzyl alcohol-1,1-h2 and -1,1-d2 by NAD+ was found to be characterized by large deuterium isotope effects (kH/kD = 4.1 plus or minus 0.1) between pH 7.5 and 9.5, indicating a rate-limiting hydride trahsfer step in this pH range; a plot of kCAT versus pH could be fit to a theoretical titration curve, pK = 8.25, where kCAT increases with increasing pH. The Michaelis constnat for p-CH3 benzyl alcohol was independent of pH. The reduction of p-CH3 benzaldehyde by NADH and reduced nicotinamide adenine dinucleotide with deuterium in the 4-A position (NADD) cound not be studied below pH 8.5 due to substrate inhibition; however, between pH 8.5 and 9.5, kCAT was found to decrease with increasing pH and to be characterized by significant isotope effects (kH/kD = 3.3 plus or minus 0.3). In the case of acetaldehyde reduction by NADH and NADD, isotope effects were found to be small and exxentially invariant (kH/kD = 2.O plus or minus 0.4) between pH 7.2 and 9.5, suggesting a partially rate-limiting hydride transger step for this substrate; a plot of kCAT/K'b (where K'b is the Michaelis constant for acetaldehyde) versus pH could be fit to a titration curve, pK = 8.25. The titration curve for acetaldehyde reduction has the same pK but is opposite in direction to that observed for p-CH3 benzyl alcohol oxidation. The data presented in this paper indicate a dependence on different enzyme forms for aldehyde reduction and alcohol oxidation and are consistent with a single active site side chain, pK = 8.25, which functions in acid-base catalysis of the hydride transfer step.