Mechanism of the reaction catalyzed by mandelate racemase. 1. Chemical and kinetic evidence for a two-base mechanism

Journal Article · Tue Sep 24 00:00:00 EDT 1991 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00102a018· OSTI ID:5822816

Powers, V M; Koo, C W; Kenyon, G L ^[1]; Gerlt, J A; Kozarich, J W ^[2]

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1. Univ. of California, San Francisco (United States)
2. Univ. of Maryland, College Park (United States)

The fate of the {alpha}-hydrogen of mandelate in the reaction catalyzed by mandelate racemase has been investigated by a mass spectroscopic method. The method entails the incubation of (R)- or (S)-({alpha}-{sup 1}H) mandelate in buffered D{sub 2}O to a low extent of turnover (about 5-8%), esterification of the resulting mixture of mandelates with diazomethane, derivatization of the methyl esters with a chiral derivatizing agent, and quantitation of the isotope content of the {alpha}-hydrogen of both substrate and product by gas chromatography/mass spectrometric analysis. No significant substrate-derived {alpha}-protium was found in the product for racemization in either direction. In addition, in the (R) to (S) direction almost no exchange of the {alpha}-hydrogen in the remaining (R) substrate pool occurred, but in the (S) to (R) direction 3.5-5.1% exchange of the {alpha}-hydrogen in the remaining substrate (after 5.1-7.2% net turnover) was found. Qualitatively similar results were obtained in the (S) to (R) direction in H{sub 2}O when (S)-({alpha}-{sup 2}H)mandelate was used as substrate. In other experiments, an overshoot in the progress curve was observed when the racemization of either enantiomer of ({alpha}-{sup 1}H) mandelate in D{sub 2}O was monitored by following the change in ellipticity of the reaction mixture; the magnitude of the overshoot was greater in the (R) to (S) than in the (S) to (R) direction. All of the available data indicate that the reaction catalyzed by mandelate racemase proceeds by a two-base mechanism, in contrast to earlier proposals.

• OSTI ID: 5822816
• Journal Information: Biochemistry; (United States), Vol. 30:38; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ISOMERASES
BIOCHEMICAL REACTION KINETICS
MANDELIC ACID
RACEMATES
BIOLOGICAL PATHWAYS
DEUTERIUM COMPOUNDS
HEAVY WATER
SUBSTRATES
CARBOXYLIC ACIDS
ENZYMES
HYDROGEN COMPOUNDS
HYDROXY ACIDS
KINETICS
ORGANIC ACIDS
ORGANIC COMPOUNDS
OXYGEN COMPOUNDS
PROTEINS
REACTION KINETICS
WATER
550200* - Biochemistry