Title

Unraveling the Catalytic Mechanism of Nitrile Hydratases

Document Type

Article

Publication Date

3-9-2007

Source Publication

Journal of Biological Chemistry

Source ISSN

0021-9258

Abstract

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants kcat and Km 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 pKES1 and pKES2 values of 5.9 ± 0.1 and 9.2 ± 0.1 (kcat′ = 130 ± 1 s-1), respectively, and pKE1 and pKE2 values of 5.8 ± 0.1 and 9.1 ± 0.1 (kcat′/Km′ = (6.5 ± 0.1) × 103 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(kcat) versus 1/T, providing Ea = 23.0 ± 1.2 kJ/mol. The thermal stability of PtNHase also allowed ΔH0 ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pKES1 and pKES2 values. Based on ΔH0ion data, pKES1 is assigned to βTyr68, whereas pKES2 is assigned to βArg52, βArg157, or αSer112 (NHases are α2β2-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.

Comments

Journal of Biological Chemistry, Vol. 282, No. 10 (March 9, 2007): 7397-7404. DOI.

Richard Holz was affiliated with the Loyola University of Chicago at the time of publication.