Mechanism of urocanase as studied by deuterium isotope effects and labeling patterns

Abstract

Nicotinamide adenine dinucleotide (NAD) dependent urocanase (4'-imidazolone-5'-propionate hydro-lyase, EC 4.2.1.49) from Pseudomonas putida was found to catalyze an exchange reaction between solvent and the 4'-hydrogen of urocanate or imidazolepropionate at a rate faster than that of overall deuterium was compared to unlabeled urocanate as a substrate, no isotope rate effect was noted. For examination of the possibility of an NAD+-mediated intramolecular hydride transfer of the 4'-hydrogen to a position on the side chain of oxoimidazolepropionate, the origins of hydrogen at positions 2 and 3 in the propionate chain were studied as a function of reaction time and extent of exchange of the 4'-hydrogen. No transfer of hydrogen from the 4' position to the side chain was observed, thereby eliminating mechanisms requiring hydride transfer via NADH between these positions. Catalytic rates in 1H2O vs. 2H2O revealed a 3-fold difference which was ascribed to a rate-limiting proton addition step. Similarly, a 5-fold decrease in Vmax was found for the reverse reaction when oxoimidazole[2,3-2H2]propionate was compared to unlabeled oxoimidazolepropionate. These data support a mechanism involving water addition across the conjugated double bond system of urocanate, rather than an internal oxidation--reduction process, yet NAD+ is required. A mechanism is proposed which uses electron delocalization in the imidazole nucleus, via an imidazole--NAD adduct, to facilitate water attack and subsequent formation of oxoimidazolepropionate.

A covalent NAD intermediate in the urocanase reaction

“…The isolation and characterization of a covalent adduct between the NAD from urocanase and imidazolepropionate support a chemistry for this reaction distinct from that for an NADH-mediated transfer of a hydride ion. While novel mechanisms for urocanase catalysis have been previously proposed in which the coenzyme acts as an electrophile, forming a covalent addition complex with the substrate to facilitate electron delocalization (Egan et al, 1981;Keul et al, 1979), the present report constitutes the first direct support for such a possibility. Our results, although derived from the study of a substrate analogue rather than the actual substrate, would seem relevant to the events occurring on the enzyme upon binding urocanate since recent reports indicate that urocanase catalyzes a solvent exchange of the 5 hydrogen of both imidazolepropionate and urocanate (Egan et al, 1981; Gerlinger & Retey, 1980).…”

“…While novel mechanisms for urocanase catalysis have been previously proposed in which the coenzyme acts as an electrophile, forming a covalent addition complex with the substrate to facilitate electron delocalization (Egan et al, 1981;Keul et al, 1979), the present report constitutes the first direct support for such a possibility. Our results, although derived from the study of a substrate analogue rather than the actual substrate, would seem relevant to the events occurring on the enzyme upon binding urocanate since recent reports indicate that urocanase catalyzes a solvent exchange of the 5 hydrogen of both imidazolepropionate and urocanate (Egan et al, 1981; Gerlinger & Retey, 1980). In addition, the following paper (Matherly et al, 1982) presents kinetic evidence for the existence of a reaction intermediate involving NAD and urocanate or imidazolepropionate.…”

“…Although imidazolepropionate is not hydrated by urocanase, both the analogue and urocanate undergo an enzyme-catalyzed exchange of the imidazole 5 hydrogen with solvent (Egan et al, 1981;Gerlinger & Retey, 1980). This would imply a similar mode of binding for the two compounds and suggests that a study of the chromophore produced upon imidazolepropionate binding might provide new mechanistic information regarding urocanase catalysis.…”

Roles of cysteine sulfinate and transaminase on in vitro dark reversion of urocanase in Pseudomonas putida

“…The dependence of the catalytic activity of Pseudomonas putida urocanase on added NAD+ has been demonstrated previously (16). The mechanism of this unique enzymatic reaction is not yet clear, but a new proposal for the role of NAD+ was offered by Egan et al (3). Urocanase from P. putida is almost completely inactivated in cold, starved cells (9) and partially inactivated in commercially or laboratory-grown cells (12).…”

Transient-state kinetic analysis of urocanase

“…The mechanism for the urocanase-catalyzed conversion of urocanic acid to 5-hydroxyimidazolepropionic acid, the enol form of oxoimidazolepropionic acid, has been the focus of a number of studies aimed at understanding the role of NAD+ (nicotinamide adenine dinucleotide) in the reaction process. While urocanase (EC 4.2.1.49) has an absolute catalytic requirement for a tightly bound NAD+ (Egan & Phillips, 1977;Keul et al, 1979), NADH (reduced NAD) is apparently not formed during catalysis, as shown by the absence of a direct transfer of a hydride ion in deuterium labeling experiments (Egan et al, 1981) and by the demonstration that the enol tautomer of oxoimidazolepropionic acid is the reaction product (Kaeppeli & Retey, 1971; .…”