scholarly journals Kinetic Mechanism of Human dUTPase, an Essential Nucleotide Pyrophosphatase Enzyme

2007 ◽  
Vol 282 (46) ◽  
pp. 33572-33582 ◽  
Author(s):  
Judit Tóth ◽  
Balázs Varga ◽  
Mihály Kovács ◽  
András Málnási-Csizmadia ◽  
Beáta G. Vértessy
Keyword(s):  
Steady State ◽  
Active Site ◽  
Reaction Pathway ◽  
Three Dimensional ◽  
Kinetic Mechanism ◽  
Dimensional Structure ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Novel Target ◽  
Rate Limiting

Human dUTPase is essential in controlling relative cellular levels of dTTP/dUTP, both of which can be incorporated into DNA. The nuclear isoform of the enzyme has been proposed as a promising novel target for anticancer chemotherapeutic strategies. The recently determined three-dimensional structure of this protein in complex with an isosteric substrate analogue allowed in-depth structural characterization of the active site. However, fundamental steps of the dUTPase enzymatic cycle have not yet been revealed. This knowledge is indispensable for a functional understanding of the molecular mechanism and can also contribute to the design of potential antagonists. Here we present detailed pre-steady-state and steady-state kinetic investigations using a single tryptophan fluorophore engineered into the active site of human dUTPase. This sensor allowed distinction of the apoenzyme, enzyme-substrate, and enzyme-product complexes. We show that the dUTP hydrolysis cycle consists of at least four distinct enzymatic steps: (i) fast substrate binding, (ii) isomerization of the enzyme-substrate complex into the catalytically competent conformation, (iii) a hydrolysis (chemical) step, and (iv) rapid, nonordered release of the products. Independent quenched-flow experiments indicate that the chemical step is the rate-limiting step of the enzymatic cycle. To follow the reaction in the quenched-flow, we devised a novel method to synthesize γ-32P-labeled dUTP. We also determined by indicator-based rapid kinetic assays that proton release is concomitant with the rate-limiting hydrolysis step. Our results led to a quantitative kinetic model of the human dUTPase catalytic cycle and to direct assessment of relative flexibilities of the C-terminal arm, critical for enzyme activity, in the enzyme-ligand complexes along the reaction pathway.

scholarly journals The Three-Dimensional Structure of the Titanium-Centered Active Site during Steady-State Catalytic Epoxidation of Alkenes

2003 ◽  
Vol 107 (8) ◽  
pp. 1932-1932 ◽  
Author(s):  
Gopinathan Sankar ◽  
John Meuring Thomas ◽  
C. Richard A. Catlow ◽  
Carolyn M. Barker ◽  
David Gleeson ◽  
...  
Keyword(s):  
Steady State ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Catalytic Epoxidation ◽  
Epoxidation Of Alkenes

scholarly journals The effects of hydrogen ion concentration on the simplest steady-state enzyme systems

1971 ◽  
Vol 123 (3) ◽  
pp. 445-453 ◽  
Author(s):  
P. Ottolenghi
Keyword(s):  
Steady State ◽  
Active Site ◽  
Hydrogen Ion ◽  
Ion Concentration ◽  
Hydrogen Ion Concentration ◽  
Substrate Complex ◽  
Perturbation Term ◽  
Enzyme Substrate ◽  
Enzyme Systems ◽  
Steady State Kinetics

Laidler (1955) showed that consideration of the effect of pH on enzymic mechanisms that obey steady-state kinetics leads to the inclusion in the equations of a ‘perturbation term’ that can introduce curvature into the Lineweaver–Burk plots. He also stated conditions in which this term vanishes. This term can lead to apparent activation by substrate. Further, several cases are shown in which simplification, but not disappearance, of the perturbation term can lead to linearity of Lineweaver–Burk plots. These cases arise when the ionization of groups at the active site either is unaffected or is completely prevented when the enzyme–substrate complex is formed. It is also shown that V(app.) can vary with pH without a concomitant change in Km(app.) in certain cases that obey steady-state kinetics without implying that Km=Ks. When the perturbation term is significant, Dixon's (1953) rules for the calculation of pK values will not always apply.

scholarly journals Reductive half-reaction of the H172Q mutant of trimethylamine dehydrogenase: evidence against a carbanion mechanism and assignment of kinetically influential ionizations in the enzyme–substrate complex

1999 ◽  
Vol 341 (2) ◽  
pp. 307-314 ◽  
Author(s):  
Jaswir BASRAN ◽  
Michael J. SUTCLIFFE ◽  
Russ HILLE ◽  
Nigel S. SCRUTTON
Keyword(s):  
Active Site ◽  
Bond Cleavage ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Trimethylamine Dehydrogenase ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Ph Range ◽  
Rate Limiting ◽  
Macroscopic Pka

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172 Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a ‘carbanion’ mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5±0.2 and 8.4±0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4±0.1 and 10.5±0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4±0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

The Three-Dimensional Structure of the Titanium-Centered Active Site during Steady-State Catalytic Epoxidation of Alkenes

2001 ◽  
Vol 105 (38) ◽  
pp. 9028-9030 ◽  
Author(s):  
Gopinathan Sankar ◽  
John Meurig Thomas ◽  
C. Richard A. Catlow ◽  
Carolyn M. Barker ◽  
David Gleeson ◽  
...  
Keyword(s):  
Steady State ◽  
Active Site ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure ◽  
Catalytic Epoxidation ◽  
Epoxidation Of Alkenes

scholarly journals Kinetic studies of the polygalacturonase enzyme from Colletotrichum lindemuthianum

1992 ◽  
Vol 285 (2) ◽  
pp. 551-556 ◽  
Author(s):  
G Waksman ◽  
G Turner ◽  
A R Walmsley
Keyword(s):  
Steady State ◽  
Fluorescence Intensity ◽  
Kinetic Studies ◽  
Kinetic Mechanism ◽  
Colletotrichum Lindemuthianum ◽  
Protein Fluorescence ◽  
Substrate Complex ◽  
Single Turnover ◽  
Enzyme Substrate ◽  
Rapid Quench

The intrinsic protein fluorescence of the polygalacturonase from Colletotrichium lindemuthianum was exploited in stopped-flow experiments aimed at elucidating the kinetic mechanism for this enzyme. Binding of the polymeric substrate polygalacturonic acid (PGA) essentially produced a triphasic fluorescence profile. There was an initial rapid quench in fluorescence, consistent with the rapid formation of the enzyme-substrate complex, with an equilibrium constant of about 8 x 10(-4)% (w/v) PGA (about 0.27 microM). There then followed a near-constant fluorescence phase, attributable to turnover of the enzyme-substrate complex as a steady-state intermediate. As the concentration of the steady-state intermediate became depleted, towards the end of the reaction, there was a partial return of the fluorescence intensity. This phase is attributed to a final, single turnover of the enzyme at the end of the reaction. The fluorescence intensity does not return to its original level due to product remaining bound at the end of the reaction.

scholarly journals Imaging active site chemistry and protonation states: NMR crystallography of the tryptophan synthase α-aminoacrylate intermediate

2022 ◽  
Vol 119 (2) ◽  
pp. e2109235119
Author(s):  
Jacob B. Holmes ◽  
Viktoriia Liu ◽  
Bethany G. Caulkins ◽  
Eduardo Hilario ◽  
Rittik K. Ghosh ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Reaction Pathway ◽  
Three Dimensional ◽  
Integrated Approach ◽  
Nucleophilic Attack ◽  
Dimensional Structure ◽  
Tryptophan Synthase ◽  
Hydrogen Bond Donor ◽  
Nmr Crystallography

NMR-assisted crystallography—the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry—holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5′-phosphate–dependent enzymes that catalyze β-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate β-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid–base catalytic residue βLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cβ and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.

Effect of Modifiers on the Hydrolysis of Basic and Neutral Peptides by Carboxypeptidase B

1975 ◽  
Vol 53 (7) ◽  
pp. 747-757 ◽  
Author(s):  
Graham J. Moore ◽  
N. Leo Benoiton
Keyword(s):  
Active Site ◽  
Active Sites ◽  
Kinetic Behavior ◽  
Carboxypeptidase A ◽  
Phenyllactic Acid ◽  
Carboxypeptidase B ◽  
Substrate Complex ◽  
Enzyme Substrate ◽  
Basic Peptides ◽  
Hydrolysis Of

The initial rates of hydrolysis of Bz-Gly-Lys and Bz-Gly-Phe by carboxypeptidase B (CPB) are increased in the presence of the modifiers β-phenylpropionic acid, cyclohexanol, Bz-Gly, and Bz-Gly-Gly. The hydrolysis of the tripeptide Bz-Gly-Gly-Phe is also activated by Bz-Gly and Bz-Gly-Gly, but none of these modifiers activate the hydrolysis of Bz-Gly-Gly-Lys, Z-Leu-Ala-Phe, or Bz-Gly-phenyllactic acid by CPB. All modifiers except cyclohexanol display inhibitory modes of binding when present in high concentration.Examination of Lineweaver–Burk plots in the presence of fixed concentrations of Bz-Gly has shown that activation of the hydrolysis of neutral and basic peptides by CPB, as reflected in the values of the extrapolated parameters, Km(app) and keat, occurs by different mechanisms. For Bz-Gly-Gly-Phe, activation occurs because the enzyme–modifier complex has a higher affinity than the free enzyme for the substrate, whereas activation of the hydrolysis of Bz-Gly-Lys derives from an increase in the rate of breakdown of the enzyme–substrate complex to give products.Cyclohexanol differs from Bz-Gly and Bz-Gly-Gly in that it displays no inhibitory mode of binding with any of the substrates examined, activates only the hydrolysis of dipeptides by CPB, and has a greater effect on the hydrolysis of the basic dipeptide than on the neutral dipeptide. Moreover, when Bz-Gly-Lys is the substrate, cyclohexanol activates its hydrolysis by CPB by increasing both the enzyme–substrate binding affinity and the rate of the catalytic step, an effect different from that observed when Bz-Gly is the modifier.The anomalous kinetic behavior of CPB is remarkably similar to that of carboxypeptidase A, and is a good indication that both enzymes have very similar structures in and around their respective active sites. A binding site for activator molecules down the cleft of the active site is proposed for CPB to explain the observed kinetic behavior.

THEORY OF THE TRANSIENT PHASE IN AN ENZYME SYSTEM INVOLVING TWO ENZYME-SUBSTRATE COMPLEXES: THE CASE OF THE FORMATION OF PRODUCTS FROM THE FIRST COMPLEX

1959 ◽  
Vol 37 (4) ◽  
pp. 737-743 ◽  
Author(s):  
Ludovic Ouellet ◽  
James A. Stewart
Keyword(s):  
Experimental Data ◽  
Steady State ◽  
Active Site ◽  
Substrate Concentration ◽  
Enzyme System ◽  
Kinetic Scheme ◽  
Enzyme Concentration ◽  
Theoretical Treatment ◽  
Transient Phase ◽  
Enzyme Substrate

A theoretical treatment is worked out for the kinetic scheme[Formula: see text]in which the concentration of P1 is followed. The steady-state and transient phase equations are obtained subject to the condition that the substrate concentration is greatly in excess of the enzyme concentration. The conditions under which evidence in favor of this mechanism can be obtained from experimental data are discussed. Under certain conditions, the weight of the enzyme corresponding to one active site can be determined. Methods for the evaluation of the different constants are described.

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