Diacetyl (Acetoin) Reductase from Aerobacter aerogenes

Abstract

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...

Purification and Properties of a Diacetyl Reductase from Escherichia coli

“…8, insert) yielded the kinetic parameters (K, Vmax) for the diacetyl-dependent oxidation of NADPH ( Table 3). The Km value for diacetyl was found to be 4.44 mM, a value which is of the same order of magnitude as the Km obtained with the diacetyl (acetoin) reductase of A. aerogenes (13). Additionally, the kinetic constants obtained with ethyl acetoacetate and acetoacetyl N-acetylcysteamine as substrates are listed in Table 3.…”

Purification and characterization of diacetyl-reducing enzymes from Staphylococcus aureus

“…pH-activity profiles and affinity for the coenzyme and substrates K, values for the carbonyl substrate between 1.2 and 30 mm and for NADH between 4 /M and 0.14 mm have been reported for NAD-specific enzymes that reduce diacetyl (Branen & Keenan, 1970;Johansen et al, 1973;Larsen et al, 1973;Louis-Eugene et al, 1984). Our data for enzyme B remain in an intermediate position between these values.…”

Formation of 2,3‐pentanediol from 2,3‐pentanedione and acetylethylcarbinol by diacetyl(acetoin)reductase from Aerobacter aerogenes. A possible new pathway

“…The enzyme has been characterized kinetically and found to follow an ordered bi-bi mechanism [3,4] .…”