Protonic reorganization and substrate structure in catalysis by serine proteases

Elrod, James P.; Hogg, John L.; Quinn, Daniel M.; Venkatasubban, K. S.; Schowen, Richard L.

doi:10.1021/ja00531a039

Cited by 69 publications

(49 citation statements)

References 2 publications

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“…atom fraction of 0.5 (33,34), calculation of a midpoint partial solvent isotope effect often helps in determining the number of protons involved in the catalytic reaction. Equations 5-7, derived by Elrod et al (33), allowed the calculation of midpoint partial solvent isotope effects when the experimental data were obtained at different atom fractions,…”

Section: Catalytically Important Residues In Nitrile Hydratasesmentioning

confidence: 99%

See 1 more Smart Citation

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Mitra

Holz

2007

Journal of Biological Chemistry

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To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k cat and K m for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK ES1 and pK ES2 values of 5.9 ؎ 0.1 and 9.2 ؎ 0.1 (k cat ‫؍‬ 130 ؎ 1 s ؊1 ), respectively, and pK E1 and pK E2 values of 5.8 ؎ 0.1 and 9.1 ؎ 0.1 (k cat /K m ‫؍‬ (6.5 ؎ 0.1) ؋ 10 3 s ؊1 mM ؊1 ), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60°C, an Arrhenius plot was constructed by plotting ln(k cat ) versus 1/T, providing E a ‫؍‬ 23.0 ؎ 1.2 kJ/mol. The thermal stability of PtNHase also allowed ⌬H 0 ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK ES1 and pK ES2 values. Based on ⌬H ion 0 data, pK ES1 is assigned to ␤Tyr 68 , whereas pK ES2 is assigned to ␤Arg 52 , ␤Arg 157 , or ␣Ser 112 (NHases are ␣ 2 ␤ 2 -heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron-and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.Nitrile hydratase (NHase 2 ; EC 4.2.1.84), one of the enzymes in the nitrile degradation pathway, catalyzes the hydrolysis of nitriles to their corresponding higher value amides in a chemo-, regio-, and/or enatioselective manner at ambient pressures and temperatures at physiological pH (Scheme 1) (1-6). NHases have attracted substantial interest as biocatalysts for industrial applications such as the large-scale production of acrylamide (3, 7-9) and nicotinamide (10). Acrylamide production utilizing the bacterium Rhodococcus rhodochrous J1 has increased to Ͼ30,000 tons/year (3), whereas Ͼ3500 tons of nicotinamide are produced per year (11). Yields of Ͼ99% are achieved, and the formation of by-products such as acrylic acid, which plagues traditional methodology, is completely avoided. However, one of the most attractive features of nitrile-metabolizing enzymes is their ability to selectively hydrolyze one cyano group of a dinitrile to its corresponding amine, something that is virtually impossible using conventional chemical methods (12-14). Therefore, the potential use of nitrile-hydrolyzing enzymes for the production of several fine chemicals is increasingly recognized.NHases are metalloenzymes that contain either a non-heme Fe(III) ion (iron-type) or a non-corrin Co(III) ion (cobalt-type) in their active site and are typically ␣ 2 ␤ 2 -heterotetramers (5, 6, 15, 16). In all known NHases, each ␣-subunit has a highly homologous amino acid sequence (CXYCSCX) that forms the metal-binding site. Cobalt-type NHases contain threonine and tyrosine in the -C(T/S)YCSC(Y/T)-se...

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Section: Catalytically Important Residues In Nitrile Hydratasesmentioning

confidence: 99%

“…Equations 5-7, derived by Elrod et al (33), allowed the calculation of midpoint partial solvent isotope effects when the experimental data were obtained at different atom fractions,…”

Section: Catalytically Important Residues In Nitrile Hydratasesmentioning

confidence: 99%

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Mitra

Holz

2007

Journal of Biological Chemistry

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“…Because the largest deviation for theoretical proton inventory curves occurs at atom fractions of 0.5 (47), calculation of a mid-point partial solvent isotope effect often helps in determining the number of protons involved in the catalytic reaction. The following equations, derived by Elrod et al (47), allowed the calculation of mid-point partial solvent isotope effects when the experimental …”

Section: Kineticmentioning

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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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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“…When the tetrahedral intermediate is broken in the direction of products, the pK a of Asp"!# can return to its original value, owing to the reorganization of the hydrogen-bonded network. This model is further supported by the β-#H secondaryisotope effect, the kinetic #H # O solvent-isotope effect and protoninventory results obtained with serine proteases [26,27]. Substrates that lack most of the structural features of a natural substrate and can be considered only little or not at all to affect the enzyme conformation, exhibit one-proton catalysis, whereas more specific substrates result in two-proton (or multiple-proton) catalysis.…”

Section: Enzyme-substrate Interaction In the Catalytic Triad Of Serinmentioning

confidence: 68%

“…This suggests an increase in the pK a of Asp"!# during the reaction. In the following discussion I will show experimental evidence for the change in the pK a of Asp"!# by reinterpreting previous NMR [5,6] and isotope-effect [26,27] studies. pK a adjustment has been previously proposed for ionizing groups of serine proteases [8,16,[28][29][30][31], but direct experimental evidence for a change in the pK a of Asp"!# has not been described.…”

Section: Enzyme-substrate Interaction In the Catalytic Triad Of Serinmentioning

confidence: 91%

Enzyme–substrate interaction in the catalytic triad of serine proteases: increase in the pKa of Asp102

Neuvonen

1997

Biochemical Journal

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Protonic reorganization and substrate structure in catalysis by serine proteases

Cited by 69 publications

References 2 publications

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Unraveling the Catalytic Mechanism of Nitrile Hydratases

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

Enzyme–substrate interaction in the catalytic triad of serine proteases: increase in the pKa of Asp102

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