No abstract

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...

Mitochondrial Grx2 is a new member of the thioredoxin superfamily that has been found to bind a [2Fe-2S] cluster in a novel coordination motif at the interface of a homodimer, where cluster binding occurs via a catalytic cysteine residue and a molecule of GSH (per monomer). The (Grx2)(2)-[2Fe-2S] dimer is thought to undergo cluster destruction and monomerization in a redox-induced pathway of activation. In this report, we make use of protein film voltammetry (PFV) as a method to probe the stability of the Grx2-[2Fe-2S] cluster, using oxidative poises of varying potential and duration to probe the thermodynamic and kinetic stability of the cluster's electrochemical response. We find that the cluster signal is stable at positive potentials up to 0.5 V but that cluster destruction occurs readily when oxidative pulses in excess of this value are applied.

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline (L-Met-p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV-Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli (EcMetAP-I) and the type II MetAP from Pyrococcus furiosus (PfMetAP-II). Using L-Met-p-NA, the kinetic constants k(cat) and K(m) were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a "standard" high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k(cat) for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that L-Met-p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.