Martin Noble is the Professor of Structural Biology and Anticancer Drug Design at the Northern In... more Martin Noble is the Professor of Structural Biology and Anticancer Drug Design at the Northern Institute for Cancer Research in Newcastle University. His research to date has focused on structural and biochemical aspects of signal transduction, especially as it applies to the regulation of the eukaryotic cell cycle. In the new setting of the NICR, his group will continue to investigate the intermolecular interactions that govern cell division, and will seek to generate small molecular modulators of those interactions as chemical probes and leads for anticancer drug design.
The article deals with the structure and function of liver alcohol dehydrogenase and reviews main... more The article deals with the structure and function of liver alcohol dehydrogenase and reviews mainly literature published after 1979, i.e., summarizes progress made in the field since Klinman presented her review on alcohol dehydrogenases. The emphasis will be on high-resolution crystallographic data, results obtained with metal-substituted enzyme derivatives, and on the mechanism and pH dependence of the catalytic reaction.
Detailed studies of chemical mechanism and transition state structure in enzyme-catalyzed reactio... more Detailed studies of chemical mechanism and transition state structure in enzyme-catalyzed reactions are frequently hampered by rate determining protein isomerization and product desorption steps. Among dehydrogenases, the alcohol dehydrogenases are almost unique in the successful kinetic isolation of the bond rearrangement step(s). Some of the pertinent mechanistic questions are (1) the mode of hydrogen activation (hydride ion vs. hydrogen atom), (2) the existence and nature of chemical intermediates, (3) a role for an active site Zn+2 vs. Zn+2-OH2 in acid-base catalysis, (4) the "concertedness" of such catalysis with heavy atom rearrangements, and (5) the extent to which the structure of the transition state resembles oxidized substrates vs. reduced products. Although definitive answers to each of these questions are not yet available, a wealth of information has been amassed for both yeast and horse liver alcohol dehydrogenase. Importantly, kinetic studies support a conservation of mechanism and transition state structure among dehydrogenases from divergent evolutionary sources.
In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneli... more In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneling, the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of benzyl alcohol was studied at sub-zero temperatures. Previous work showed that wild type HLADH has significant kinetic complexity down to -50 degrees C due to slow binding and loss of substrate [S.-C. Tsai, J.P. Klinman, Biochemistry, 40 (2001) 2303]. A strategy was therefore undertaken to reduce kinetic complexity at sub-zero temperatures, using a photolabile (caged) benzyl alcohol that prebinds to the enzyme and yields the active substrate upon photolysis. By computer modeling, a series of caged alcohols were designed de novo, synthesized, and characterized with regard to photolysis and binding properties. The o-nitrobenzyl ether 15, with a unique long tail, was found to be most ideal. At sub-zero temperatures in 50% MeOH, a two-phase kinetic trace and a rate enhancement by the use of 15 were observed. Despite the elimination of substrate binding as a rate-limiting step, the use of caged benzyl alcohol does not produce a measurable H/D kinetic isotope effect. Unexpectedly, the observed fast phase corresponds to multiple enzyme turnovers, based on the stoichiometry of the substrate to enzyme. Possible side reactions and their effects, such as the re-oxidation of bound NADH and the dissipation of photo-excitation energy, may offer an explanation for the observed multiple-turnovers. The lack of observable deuterium isotope effects offers a cautionary note for the application of caged substrates to isolate and study chemical steps of enzyme reactions, particularly when NADH is involved in the reaction pathway.
Solvent and alpha-secondary isotope effects have been measured in the yeast alcohol dehydrogenase... more Solvent and alpha-secondary isotope effects have been measured in the yeast alcohol dehydrogenase reaction, under conditions of a rate-limiting transfer of hydrogen between coenzyme and substrate. Determination of catalytic constants (at saturating concentrations of substrate and coenzyme) in H2O and D2O as a function of pH(D) has allowed the separation of solvent effects on pKa from kcat: delta pKa = pKD--pKH = 0.02--0.21, kH2O/kD2O = 1.20 +/- 0.09 in the direction of p-methoxybenzyl alcohol oxidation, and kH2O/kD2O = 0.50 +/- 0.05 and 0.58 +/- 0.06 for p-methoxybenzaldehyde reducation by NADH and [4-2H]NADH. The small effect of D2O on pKa, which contrasts with the common observation that delta pKa congruent to 0.4--0.6, is tentatively assigned to ionization of an active-site ZnOH2. The near absence of an isotope effect on kcat in the direction of alcohol oxidation rules out a mechanism involving concerted catalysis by an active-site base of hydride transfer. In the direction of aldehyde reduction, the observation of inverse isotope effects on kcat is concluded to reflect displacement of zinc-bound water by substrate to form an inner-sphere complex, subsequent to the E.S complex. Equilibrium alpha-secondary isotope effects, measured as a frame of reference for kinetic values, indicate KH/KT = 1.33 +/- 0.05 and 1.34 +/- 0.09 for the oxidation of [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol, respectively. Kinetic alpha-secondary isotope effects are within experimental error of equilibrium values, kH/kT = 1.34 +/- 0.07 and 1.38 +/- 0.02 for [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol oxidation, respectively. The near identity of kinetic and equilibrium alpha-secondary isotope effects in the direction of alcohol oxidation implicates a transition-state structure which resembles aldehyde with regard to bond hybridization properties. This result contrasts sharply with previously reported structure--reactivity correlations, which implicate a transition-state structure resembling alcohol with regard to charge properties. The significance of these findings to the mechanism of NAD(P)H-dependent redox reactions is discussed.
Copper amine oxidases (CAOs) catalyze the two-electron oxidation of primary amines to aldehydes, ... more 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.
Martin Noble is the Professor of Structural Biology and Anticancer Drug Design at the Northern In... more Martin Noble is the Professor of Structural Biology and Anticancer Drug Design at the Northern Institute for Cancer Research in Newcastle University. His research to date has focused on structural and biochemical aspects of signal transduction, especially as it applies to the regulation of the eukaryotic cell cycle. In the new setting of the NICR, his group will continue to investigate the intermolecular interactions that govern cell division, and will seek to generate small molecular modulators of those interactions as chemical probes and leads for anticancer drug design.
The article deals with the structure and function of liver alcohol dehydrogenase and reviews main... more The article deals with the structure and function of liver alcohol dehydrogenase and reviews mainly literature published after 1979, i.e., summarizes progress made in the field since Klinman presented her review on alcohol dehydrogenases. The emphasis will be on high-resolution crystallographic data, results obtained with metal-substituted enzyme derivatives, and on the mechanism and pH dependence of the catalytic reaction.
Detailed studies of chemical mechanism and transition state structure in enzyme-catalyzed reactio... more Detailed studies of chemical mechanism and transition state structure in enzyme-catalyzed reactions are frequently hampered by rate determining protein isomerization and product desorption steps. Among dehydrogenases, the alcohol dehydrogenases are almost unique in the successful kinetic isolation of the bond rearrangement step(s). Some of the pertinent mechanistic questions are (1) the mode of hydrogen activation (hydride ion vs. hydrogen atom), (2) the existence and nature of chemical intermediates, (3) a role for an active site Zn+2 vs. Zn+2-OH2 in acid-base catalysis, (4) the "concertedness" of such catalysis with heavy atom rearrangements, and (5) the extent to which the structure of the transition state resembles oxidized substrates vs. reduced products. Although definitive answers to each of these questions are not yet available, a wealth of information has been amassed for both yeast and horse liver alcohol dehydrogenase. Importantly, kinetic studies support a conservation of mechanism and transition state structure among dehydrogenases from divergent evolutionary sources.
In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneli... more In order to understand the influence of protein dynamics on enzyme catalysis and hydrogen tunneling, the horse liver alcohol dehydrogenase (HLADH) catalyzed oxidation of benzyl alcohol was studied at sub-zero temperatures. Previous work showed that wild type HLADH has significant kinetic complexity down to -50 degrees C due to slow binding and loss of substrate [S.-C. Tsai, J.P. Klinman, Biochemistry, 40 (2001) 2303]. A strategy was therefore undertaken to reduce kinetic complexity at sub-zero temperatures, using a photolabile (caged) benzyl alcohol that prebinds to the enzyme and yields the active substrate upon photolysis. By computer modeling, a series of caged alcohols were designed de novo, synthesized, and characterized with regard to photolysis and binding properties. The o-nitrobenzyl ether 15, with a unique long tail, was found to be most ideal. At sub-zero temperatures in 50% MeOH, a two-phase kinetic trace and a rate enhancement by the use of 15 were observed. Despite the elimination of substrate binding as a rate-limiting step, the use of caged benzyl alcohol does not produce a measurable H/D kinetic isotope effect. Unexpectedly, the observed fast phase corresponds to multiple enzyme turnovers, based on the stoichiometry of the substrate to enzyme. Possible side reactions and their effects, such as the re-oxidation of bound NADH and the dissipation of photo-excitation energy, may offer an explanation for the observed multiple-turnovers. The lack of observable deuterium isotope effects offers a cautionary note for the application of caged substrates to isolate and study chemical steps of enzyme reactions, particularly when NADH is involved in the reaction pathway.
Solvent and alpha-secondary isotope effects have been measured in the yeast alcohol dehydrogenase... more Solvent and alpha-secondary isotope effects have been measured in the yeast alcohol dehydrogenase reaction, under conditions of a rate-limiting transfer of hydrogen between coenzyme and substrate. Determination of catalytic constants (at saturating concentrations of substrate and coenzyme) in H2O and D2O as a function of pH(D) has allowed the separation of solvent effects on pKa from kcat: delta pKa = pKD--pKH = 0.02--0.21, kH2O/kD2O = 1.20 +/- 0.09 in the direction of p-methoxybenzyl alcohol oxidation, and kH2O/kD2O = 0.50 +/- 0.05 and 0.58 +/- 0.06 for p-methoxybenzaldehyde reducation by NADH and [4-2H]NADH. The small effect of D2O on pKa, which contrasts with the common observation that delta pKa congruent to 0.4--0.6, is tentatively assigned to ionization of an active-site ZnOH2. The near absence of an isotope effect on kcat in the direction of alcohol oxidation rules out a mechanism involving concerted catalysis by an active-site base of hydride transfer. In the direction of aldehyde reduction, the observation of inverse isotope effects on kcat is concluded to reflect displacement of zinc-bound water by substrate to form an inner-sphere complex, subsequent to the E.S complex. Equilibrium alpha-secondary isotope effects, measured as a frame of reference for kinetic values, indicate KH/KT = 1.33 +/- 0.05 and 1.34 +/- 0.09 for the oxidation of [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol, respectively. Kinetic alpha-secondary isotope effects are within experimental error of equilibrium values, kH/kT = 1.34 +/- 0.07 and 1.38 +/- 0.02 for [1(S)-3H]benzyl alcohol and p-methoxy[1(S)-3H]benzyl alcohol oxidation, respectively. The near identity of kinetic and equilibrium alpha-secondary isotope effects in the direction of alcohol oxidation implicates a transition-state structure which resembles aldehyde with regard to bond hybridization properties. This result contrasts sharply with previously reported structure--reactivity correlations, which implicate a transition-state structure resembling alcohol with regard to charge properties. The significance of these findings to the mechanism of NAD(P)H-dependent redox reactions is discussed.
Copper amine oxidases (CAOs) catalyze the two-electron oxidation of primary amines to aldehydes, ... more 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.
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Papers by Judith Klinman