The kinetics of the reversible reduction of acetoin and NADH to 2,3-butanediol andNAD by diacetyl (acetoin) reductase from Aerobacter aerogenes, have been investigated at pH 7.0, 5.8, and 4.8. The reaction follows an ordered bi-bi mechanism.Acetate acts as an inhibitor of the enzyme with respect to the substrate 2,3-butanediol. This inhibition increases for decreasing pH, indicating that it is acetic acid that is the effector.These results, in combination with earlier observations, show that high acetate concentration allows maximal 2,3-butanediol and NAD production at 5.8 from pyruvate and NADH. The kinetics of the pH 6 acetolactate-forming enzyme has been investigated [3,4]. It has a sharp pH-optimum at pH 5.8 in acetate, which also acts as an activator for the enzyme. Acetate also acts as an inducer for the three enzymes leading from pyruvate to 2,3-butanediol [2,5]. Acetolactate decarboxylase has a pH-optimum a t about 6.3 in phosphate, and gives about 75O/, of optimal activity a t yH 5.8. Acetate has no effect upon the enzyme activity [6]. This indicates that the formation of acetoin is maximally favored a t pH 5.8 in the presence of acetate.Diacetyl (acetoin) reductase has recently been purified into homogeneity [2]. The enzyme is a tetramer, consisting of four equal-sized subunits [7]. The native enzyme consists of at least 12 isoelectric species, all possessing enzyme activity [8].To gain an insight into the mechanism of diacetyl (acetoin) reductase reaction, a kinetic study was carried out on reaction 3 in Fig. 1 a t pH 5.8 in acetate, phosphate and 2-(N-morpholino)-ethane-sulfonate, pH 7.0 in phosphate and pH 4.8 in acetate and morpholinoethane sulfonate. EXPERIMENTAL PROCEDURE ChemicalsThe following chemicals were purchased from Sigma Chemical Company: NAD, NADH, NADP, and NADPH. Acetoin was obtained from KochLight, 2,3-butanediol from Fluka AG, and 24N-morpho1ino)-ethane-sulfonic acid from Calbiochem. Preparation and Assay of Diacetyl (Acetoin) ReductaseThe enzyme was purified from crude extracts of Aerobacter aerogenes [2]. For spectrophotometric determinations of enzyme activity, the Zeiss model PMQII recording spectrophotometer with the thermostated cell compartment a t 25 "C was used.Initial reaction rates were measured by observing the rate of change in the absorbance at 340nm.

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A kinetic study of diacetyl (acetoin) reductase in the reaction from diacetyl to acetoin has been performed.Product inhibition plots, similar to those obtained for the reduction of acetoin and the reverse reaction, were obtained. The kinetic mechanisms for the reactions catalyzed by the enzyme are proposed.Diacetyl (acetoin) reductase catalyzes the reduction of acetoin to 2,3-butanediol as well as the reverse reaction, and the irreversible reduction of diacetyl to acetoin [1,2]. The enzyme is a tetramer with 4 equal-sized subunits [3] and consists of several isoelectric species [4]. Diacetyl (acetoin) reductase has been characterized kinetically in the former reaction, and found to follow an ordered bi-bi mechanism [5]. It can also use the analogues 2,3-pentanedione, acetylethyl carbinol, and 2,3-pentanediol as substrates [6].The reaction from 2,3-butanediol to acetoin is inhibited by acetate, and the Km for 2,3-butanediol is found to increase 10-fold at pH 5.8 in acetate, compared with the values obtained in other buffers at the same pH [5].In the present report we have studied the reduction of diacetyl to acetoin, catalyzed by homogeneous diacetyl (acetoin) reductase purified from Aerobacter aerogenes [2], and the effect of 2,3-butanediol, NAD, and acetate. MATERIALS AND METHODSThe following chemicals were purchased from Sigma Chemical Company: NAD, NADH, and diacetyl. 2,3-Butanediol was obtained from Fluka AG, and acetoin from Koch Light Labs.The enzyme was purified as described previously [2]. Initial reaction rates were measured by observing the rate of change in absorbance a t 340 nm. The assay mixture consisted of 1.0 ml containing I00 pmoles acetate or phosphate pH 5.8. The amounts of diacetyl, 2,3-butanediol, NAD, and NADH were as described in the legends to the figures. The reaction was started by adding 55ng 7 Eur. J. Biochem., Vo1.34 enzyme, which gave a linear change in absorbance in 2 min [5]. For spectrophotometric determinations of enzyme activity, the Shimadzu multipurpose recording spectrophotometer Model MPS-BOL, with the thermostated cell compartment a t 25 "C, was used. RESULTS AND DISCUSSIONThe initial velocity studies were performed with NADH as the variable substrate in the presence of several fixed concentrations of diacetyl. As shown in Fig.1, plots of reciprocal velocity intersect in the upper left quadrant, showing that the apparent Michaelis constant for one substrate is dependent on the concentration of the other.The Michaelis constants for NADH and diacetyl were derived from the secondary plots of intercepts verms reciprocal concentrations of the other substrate, and calculated to be 0.004 and 1.2 mM, respectively.If NADH was used as the variable substrate, and NAD as the product inhibitor, the inhibition patterns were competitive (Fig. 2) indicating that NADH is first added and NAD last released from the enzyme in the reaction from diacetyl to acetoin. Since acetoin is using NADH as the second substrate, it cannot be used as a product inhibitor in order to investigate the reac...

mutants of active site residues of AnAEst were determined. While the wild-type enzyme showed highest catalytic efficiency for naphthyl esters relative to phenyl esters, the R54G mutant displayed a 2.4 fold increase in catalytic efficiency for phenyl esters over naphthyl esters. The kinetic studies in conjunction with docking studies confirm the structural role of Arg54 and other active site residues in both substrate binding and catalysis.

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