The Proton Inventory Techniqu

Venkatasubban, K. S.; Schowen, Richard L.

doi:10.3109/10409238409110268

Cited by 206 publications

(323 citation statements)

References 44 publications

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“…where X i D and X i H are the mole fractions of deuterons and protons in the ith transition or reactant state whereas the subscripts T and R refer to the transition and reactant state, respectively (45,46). At pH 10.50, the best fit obtained exhibited a straight line (Fig.…”

Section: Kineticmentioning

confidence: 67%

“…One of the fractionation factors ( T2 ϭ 0.44) is similar to that obtained at pH 10.50, consistent with the proposed proton transfer in the tetrahedral transition state . The first fractionation factor ( T1 ϭ 0.76) is characteristic of a protonoxygen bond (neutral oxygen, 0.8 -1.2) with a conventional isotope effect equal to 1 (46). It is likely that at this pH, the protonation state of Glu-151 (whose pK a in the enzyme substrate complex is 5.3) results in the observed effect.…”

Section: Temperature Dependence Of K Cat and K M For E151d-aap-mentioning

confidence: 99%

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The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Bzymek

Holz

2004

Journal of Biological Chemistry

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Glutamate 151 has been proposed to act as the general acid/base during the peptide hydrolysis reaction catalyzed by the co-catalytic metallohydrolase from Aeromonas proteolytica (AAP). However, to date, no direct evidence has been reported for the role of Glu-151 during catalytic turnover by AAP. In order to elucidate the catalytic role of Glu-151, altered AAP enzymes have been prepared in which Glu-151 has been substituted with a glutamine, an alanine, and an aspartate. The Michaelis constant (K m ) does not change upon substitution to aspartate or glutamine, but the rate of the reaction changes drastically in the following order: glutamate (100% activity), aspartate (0.05%), glutamine (0.004%), and alanine (0%). Examination of the pH dependence of the kinetic constants k cat and K m revealed a change in the pK a of a group that ionizes at pH 4.8 in recombinant leucine aminopeptidase (rAAP) to 4.2 for E151D-AAP. The remaining pK a values at 5.2, 7.5, and 9.9 do not change. Proton inventory studies indicate that one proton is transferred in the rate-limiting step of the reaction at pH 10.50 for both rAAP and E151D-AAP, but at pH 6.50 two protons and general solvation effects are responsible for the observed effects in the reaction catalyzed by rAAP and E151D-AAP, respectively. Based on these data, Glu-151 is intrinsically involved in the peptide hydrolysis reaction catalyzed by AAP and can be assigned the role of a general acid and base.

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Section: Kineticmentioning

confidence: 67%

Section: Temperature Dependence Of K Cat and K M For E151d-aap-mentioning

confidence: 99%

The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Bzymek

Holz

2004

Journal of Biological Chemistry

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“…The fractionation factor for the two-proton inventory assumes both transfers contribute equally to the isotope effect in the transition state. Both estimates of transition state fractionation factors are within the range observed for proton transfers involving oxygen or nitrogen atoms in other enzymes (38). The small magnitude of the isotope effect, confirmed in these experiments to be 2-fold, and the limited precision of our rate estimates (Ϯ5%) made it impossible to convincingly distinguish between models involving transfer of one proton or two protons (Fig.…”

Section: Resultsmentioning

confidence: 82%

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

Smith

Collins²

2007

Proc. Natl. Acad. Sci. U.S.A.

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A fast-cleaving version of the Varkud satellite ribozyme, called RG, shows an apparent cis-cleavage rate constant of 5 sec ؊1 , similar to the rates of protein enzymes that catalyze similar reactions. Here, we describe mutational, pH-rate, and kinetic solvent isotope experiments that investigate the identity and rate constant of the rate-limiting step in this reaction. Self-cleavage of RG exhibits a bell-shaped rate vs. pH profile with apparent pKas of 5.8 and 8.3, consistent with the protonation state of two nucleotides being important for the rate of cleavage. Cleavage experiments in heavy water (D2O) revealed a kinetic solvent isotope effect consistent with proton transfer in the rate-limiting step. A mutant RNA that disrupts a peripheral loop-loop interaction involved in RNA folding exhibits pH-and D2O-independent cleavage Ϸ10 3 -fold slower than wild type, suggesting that this mutant is limited by a different step than wild type. Substitution of adenosine 756 in the putative active-site loop with cytosine also decreases the cleavage rate Ϸ10 3 -fold, but the A756C mutant retains pH-and D2O-sensitivity similar to wild type, consistent with this mutant and wild type being limited by the chemical step of the reaction. These results suggest that the RG ribozyme provides a good experimental system to investigate the nature of fast, rate-limiting steps in a ribozyme cleavage reaction.kinetic solvent isotope effect ͉ kinetics ͉ Neurospora ͉ pH vs. rate S ince the discovery of RNA catalysis, several natural RNAs and many more RNA and DNA sequences obtained by in vitro selection have been shown to catalyze the cleavage and/or ligation of phosphodiester bonds, as well as other chemical activities. Recent work has begun to investigate the range of catalytic mechanisms used by ribozymes and to understand the similarities and differences with protein enzymes (1-3). The local environment of a folded RNA can shift the pK a of certain nucleobases by two or more pH units into the range where they could function as proton donors or acceptors in general acidbase catalysis, similar to histidines in their protein counterparts (4). Charged nucleobases could also participate in electrostatic stabilization in the transition state. Indeed, the bell-shaped rate vs. pH curves typical of protein enzymes that use general acid-base catalysis have been observed for certain hepatitis delta virus (HDV) ribozymes (5, 6) and Varkud satellite (VS) ribozyme (this study).Most previously characterized versions of the Neurospora VS ribozyme showed rather slow cis-or trans-cleavage apparent rate constants (k obs ) in the range of 1 min Ϫ1 or less and were only slightly affected by pH between pH 5.5 and 9.0 (7); however, a trans-ligating construct did exhibit pH dependence below pH 7.0 (8). Other observations have also raised the possibility that a protonated group could be involved in the rate-limiting step of a VS ribozyme reaction pathway. For example, Strobel and colleagues (9) observed pH-dependent rescue of a ligation reaction by using nucleo...

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“…The proton inventory of D120N did not yield a straight line. These data were fitted to the Gross-Butler equation (55) and suggested greater than two protons in flight during a rate-limiting step of the reaction. Consistent with previous work (56 -58), solvent isotope studies with nitrocefin yielded k H /k D values for all enzymes that indicate proton transfer during a nucleophilic reaction (Table I).…”

Section: Resultsmentioning

confidence: 99%

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

Garrity

Carenbauer

Herron

et al. 2004

Journal of Biological Chemistry

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Metallo-␤-lactamase L1 from Stenotrophomonas maltophilia is a dinuclear Zn(II) enzyme that contains a metal-binding aspartic acid in a position to potentially play an important role in catalysis. The presence of this metal-binding aspartic acid appears to be common to most dinuclear, metal-containing, hydrolytic enzymes; particularly those with a ␤-lactamase fold. In an effort to probe the catalytic and metal-binding role of Asp-120 in L1, three site-directed mutants (D120C, D120N, and D120S) were prepared and characterized using metal analyses, circular dichroism spectroscopy, and presteady-state and steady-state kinetics. The D120C, D120N, and D120S mutants were shown to bind 1.6 ؎ 0.2, 1.8 ؎ 0.2, and 1.1 ؎ 0.2 mol of Zn(II) per monomer, respectively. The mutants exhibited 10-to 1000-fold drops in k cat values as compared with wild-type L1, and a general trend of activity, wild-type > D120N > D120C and D120S, was observed for all substrates tested. Solvent isotope and pH dependence studies indicate one or more protons in flight, with pK a values outside the range of pH 5-10 (except D120N), during a rate-limiting step for all the enzymes. These data demonstrate that Asp-120 is crucial for L1 to bind its full complement of Zn(II) and subsequently for proper substrate binding to the enzyme. This work also confirms that Asp-120 plays a significant role in catalysis, presumably via hydrogen bonding with water, assisting in formation of the bridging hydroxide/water, and a rate-limiting proton transfer in the hydrolysis reaction.

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The Proton Inventory Techniqu

Cited by 206 publications

References 44 publications

The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

The Catalytic Role of Glutamate 151 in the Leucine Aminopeptidase from Aeromonas proteolytica

Evidence for proton transfer in the rate-limiting step of a fast-cleaving Varkud satellite ribozyme

Metal Binding Asp-120 in Metallo-β-lactamase L1 from Stenotrophomonas maltophilia Plays a Crucial Role in Catalysis

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