Purification and mechanism of action of proline racemase

Abstract

The structures of inhibitors and the substrate suggest the possible involvement of a metal ion at the active center. The enzyme is a sulfhydryl enzyme and is inactive in the oxidized form. It is rapidly activated by reducing agents such as mercaptans or NaBH,.When the racemization of L-proline is carried out in D20, the optical rotation is initially negative. As the reaction proceeds, it becomes positive, and finally zero when equilibrium is reached. This "overshoot" is attributed to a primary isotope effect. sidered for the hydrogen-transfer process: a "onehydrogen-acceptor" and a "two-hydrogen-acceptor" mechanism. In the first mechanism hydrogen is transferred from the substrate to a hydrogen acceptor at the active site with the concomitant formation of an intermediate derived from the substrate. The intermediate could be a carbanion or an oxidized form of the substrate depending upon the chemical mechanism by which the a-carbon-hydrogen bond is broken. In a subsequent step hydrogen is transferred from the acceptor to the intermediate so that either the original substrate isomer or the product isomer is formed. If a single hydrogen-acceptor site participates, it must be flexible enough to interact with the a-hydrogen of both isomers, or the substrate must change its position relative to the hydrogen acceptor during the course of the racemization. The alternative mechanism is the "twohydrogen-acceptor" mechanism. Here two equivalent sites are involved. The sites are so located that one interacts with the a-hydrogen of one substrate-isomer and the other site with the a-hydrogen of the other isomer. Inversion of configuration is achieved by transferring the a-hydrogen from the substrate to one of the acceptor sites and hydrogen from the other acceptor site to the substrate-derived intermediate. The question concerning the number of hydrogen acceptor sites has not been experimentally investigated for any amino acid racemase. The general problem has been considered by Rose (1966) for enzymes which carry out proton shifts.

Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by Agrobacterium tumefaciens BY6 Requires an Epimerase and a Stereoselective C-N Lyase

“…Since the spectrum of the purified IDS-epimerase shows no significant absorption in the range of 350 to 450 nm, there is no indication of PLP as a cofactor. Many other amino acid epimerases or racemases such as diaminopimelic acid epimerase (23), 4-hydroxyproline epimerase (9), proline racemase (6,21), aspartate racemase (27), and glutamate racemase (13) also require no cofactor. We assume that enzyme-catalyzed epimerization of IDS is accomplished by a proton abstraction and subsequent return by a two-base mechanism, as proposed for PLP-independent epimerases and racemases (21,23,27).…”

“…We assume that enzyme-catalyzed epimerization of IDS is accomplished by a proton abstraction and subsequent return by a two-base mechanism, as proposed for PLP-independent epimerases and racemases (21,23,27). In a two-base mechanism, one enzyme base removes the proton from the substrate, and the conjugate base delivers a proton in the opposite direction (6).…”

Inhibition of Escherichia coli growth and diaminopimelic acid epimerase by 3-chlorodiaminopimelic acid

“…5). Analogs of proline that are planar at C-2, the normal site of epimerization, are transition state analogs and are inhibitors of proline racemase (3). By analogy, we propose that the elimination of HCl from 3-chloro-DAP results in the formation of a transition state analog that is also planar at C-2 (Fig.…”

“…Instead, the epimerization reactions for these two enzymes are characterized by proton abstraction on one face with concomitant proton donation on the opposite face as indicated in Fig. 5 (3,15,20). The transition state for this reaction is planar at the epimerized carbon (Fig.…”

Cloning and Expression inEscherichia coliof the Glutamate Racemase Gene fromPediococcus pentosaceus

“…There seems to be no possibility, therefore, that glutamate racemase could be an FAD-or pyridoxal 5I -phosphate-dependent enzyme. Thus, glutamate racemase is analogous to diaminopimelate epimerase 11 ) and proline racemase 29 ) in dispensability of cofactors. However, our preliminary observation that the proton translocation from the substrate to product is mediated by a single-base mechanism suggests a difference from these The absorption spectrum (A) was taken at 0.8 mg/ml of the enzyme in 50 mM Tris-HCI buffer (pH 7.4) with a Union Giken SM401 recording spectrophotometer.…”