Acetohydroxyacid synthase isozyme I from Escherichia coli has unique catalytic and regulatory properties

AHAS I is an isozyme of acetohydroxyacid synthase which is apparently unique to enterobacteria. It has been known for over 20 years that it has many properties which are quite different from those of the other two enterobacterial AHASs isozymes, as well as from those of “typical” AHASs which are single enzymes in a given organism. These include a unique mechanism for regulation of expression and the absence of a preference for forming acetohydroxybutyrate. We have cloned the two subunits, ilvB and ilvN, of this Escherichia coli isoenzyme and examined the enzymatic properties of the purified holoenzyme and the enzyme reconstituted from purified subunits. Unlike other AHASs, AHAS I demonstrates cooperative feedback inhibition by valine, and the kinetics fit closely to an exclusive binding model. The formation of acetolactate by AHAS I is readily reversible and acetolactate can act as substrate for alternative AHAS I-catalyzed reactions.     

Many of the functional differences between acetohydroxyacid synthase (AHAS) isozyme I and other AHASs are a result of the rapid formation and breakdown of the covalent acetolactate-thiamin diphosphate adduct in AHAS I

Acetohydroxy acid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate (ThDP)-dependent decarboxylase-ligase that catalyzes the first common step in the biosynthesis of branched-chain amino acids. In the first stage of the reaction, pyruvate is decarboxylated and the reactive intermediate hydroxyethyl-ThDP carbanion/enamine is formed. In the second stage, the intermediate is ligated to another 2-ketoacid to form either acetolactate or acetohydroxybutyrate. AHAS isozyme I from Escherichia coli is unique among the AHAS isozymes in that it is not specific for 2-ketobutyrate (2-KB) over pyruvate as an acceptor substrate. It also appears to have a different mechanism for inhibition by valine than does AHAS III from E. coli. An investigation of this enzyme by directed mutagenesis and knowledge of detailed kinetics using the rapid mixing-quench NMR method or stopped-flow spectroscopy, as well as the use of alternative substrates, suggests that two residues determine most of the unique properties of AHAS I. Gln480 and Met476 in AHAS I replace the Trp and Leu residues conserved in other AHASs and lead to accelerated ligation and product release steps. This difference in kinetics accounts for the unique specificity, reversibility and allosteric response of AHAS I. The rate of decarboxylation of the initially formed 2-lactyl-ThDP intermediate is, in some AHAS I mutants, different for the alternative acceptors pyruvate and 2-KB, putting into question whether AHAS operates via a pure ping-pong mechanism. This finding might be compatible with a concerted mechanism (i.e. the formation of a ternary donor-acceptor:enzyme complex followed by covalent, ThDP-promoted catalysis with concerted decarboxylation-carboligation). It might alternatively be explained by an allosteric interaction between the multiple catalytic sites in AHAS.     

Allosteric regulation in Acetohydroxyacid Synthases (AHASs)--different structures and kinetic behavior in isozymes in the same organisms

Acetohydroxyacid Synthases (AHASs) have separate small regulatory subunits which specifically activate the catalytic subunits with which they are associated. The binding sites for the inhibitory amino acid(s) (valine or leucine) are in the interface between two ACT (small ligand binding) domains, and are apparently found in all AHAS regulatory subunits. However, the structures and the kinetic mechanisms of the different enzymes are very heterogeneous. Among the three isozymes encoded in the enterobacteria, the regulatory patterns are different, and their different responses to the inhibitory end product valine can be rationalized, at least in part, on the basis of the regulatory subunit structures and differences in catalytic mechanisms. The regulatory subunits in "typical" single AHASs found in other bacteria are similar to that of Escherichia coli isozyme AHAS III. Eukaryotic AHASs have more complex regulatory mechanisms and larger regulatory subunits. Such AHASs have two separate ACT sequence domains on the same regulatory polypeptide and can simultaneously bind two amino acids with synergistic effects. Yeast and fungal AHASs have ATP-binding sequence inserts in their regulatory subunits and are activated by MgATP in addition to being inhibited by valine.     

Properties of subcloned subunits of bacterial acetohydroxy acid synthases.

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.     

Binding and activation of thiamin diphosphate in acetohydroxyacid synthase

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.     

The acetolactate synthase isoenzymes of Escherichia coli K-12.

Summary Strains of Escherichia coli K-12 possessing only one of the three genes coding for acetolactate synthetase activity present either in the wild type or in its ilv0603 derivative were prepared and analyzed. Extracts prepared from these strains show different values of acetolactate synthase specific activity and different sensitivity to valine inhibition. These strains show a unique pattern of growth inhibition by different substances.Temperature sensitive (ts) mutations in the ilvB and ilvG genes, have been isolated and characterized. Extracts of these strains were found to have an acetolactate synthase activity more heat labile than that of a strain containing the corresponding wild type allele. We conclude that ilvB and ilvG are the structural genes for two different forms of acetolactate synthase activity, most likely two isoenzymes. Moreover, since the strains containing a ts mutation show a temperature sensitive auxotrophy for isoleucine and valine, these two acetolactate synthases participate in isoleucine and valine biosynthesis. Similar evidence for a third acetolactate synthase, the product of the ilvHI genes, has been reported previously.We propose the following names for the acetolactate synthase isoenzymes: acetolactate synthase I (AHAS I), the product of the ilvB gene; acetolactate synthase II (AHAS II), the product of ilvG gene; and acetolactate synthase III (AHAS III), the product of the ilvHI genes.     

Acetohydroxyacid synthase: a proposed structure for regulatory subunits supported by evidence from mutagenesis

Valine inhibition of acetohydroxyacid synthase (AHAS) plays an important role in regulation of biosynthesis of branched-chain amino acids in bacteria. Bacterial AHASs are composed of separate catalytic and regulatory subunits; while the catalytic subunits appear to be homologous with several other thiamin diphosphate-dependent enzymes, there has been no model for the structure of the small, regulatory subunits (SSUs). AHAS III is one of three isozymes in Escherichia coli. Its large subunit (encoded by ilvI) by itself has 3-5 % activity of the holoenzyme and is not sensitive to inhibition by valine. The SSU (encoded by ilvH) associates with the large subunit and is required for full catalytic activity and valine sensitivity. The isolated SSU binds valine. The properties of several mutant SSUs shed light on the relation between their structure and regulatory function. Three mutant SSUs were obtained from spontaneous Val(R) bacterial mutants and three more were designed on the basis of an alignment of SSU sequences from valine-sensitive and resistant isozymes, or consideration of the molecular model developed here. Mutant SSUs N11A, G14D, N29H and A36V, when reconstituted with wild-type large subunit, lead to a holoenzyme with drastically reduced valine sensitivity, but with a specific activity similar to that of the wild-type. The isolated G14D and N29H subunits do not bind valine. Mutant Q59L leads to a valine-sensitive holoenzyme and isolated Q59L binds valine. T34I has an intermediate valine sensitivity. The effects of mutations on the affinity of the large subunits for SSUs also vary. D. Fischer's hybrid fold prediction method suggested a fold similarity between the N terminus of the ilvH product and the C-terminal regulatory domain of 3-phosphoglycerate dehydrogenase. On the basis of this prediction, together with the properties of the mutants, a model for the structure of the AHAS SSUs and the location of the valine-binding sites can be proposed.     

Subunit association in acetohydroxy acid synthase isozyme III.

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.     

Alkylation of acetohydroxyacid synthase I from Escherichia coli K-12 by 3-bromopyruvate: evidence for a single active site catalyzing acetolactate and acetohydroxybutyrate synthesis.

Acetohydroxyacid synthase I (AHAS I) purified from Escherichia coli K-12 was irreversibly inactivated by incubation with 3-bromopyruvate. Inactivation was specific, insofar as bromoacetate and iodoacetate were much less effective than bromopyruvate. Inactivation was accompanied by incorporation of radioactivity from 3-bromo[2-14C]pyruvate into acid-insoluble material. More than 95% of the incorporated radioactivity coelectrophoresed with the 60-kilodalton IlvB subunit of the enzyme through a sodium dodecyl sulfate-polyacrylamide gel; less than 5% coelectrophoresed with the 11.2-kilodalton IlvN subunit. The stoichiometry of incorporation at nearly complete inactivation was 1 mol of 14C per mol of IlvB polypeptide. These data indicate that bromopyruvate inactivates AHAS I by alkylating an amino acid at or near a single active site located in the IlvB subunit of the enzyme. We confirmed that this alkylation inactivated both AHAS reactions normally catalyzed by AHAS I. These results provide the first direct evidence that AHAS I catalyzes both acetohydroxybutyrate and acetolactate synthesis from the same active site.     

Pyruvate decarboxylase activity of the acetohydroxyacid synthase of Thermotoga maritima

Acetohydroxyacid synthase (AHAS) catalyzes the production of acetolactate from pyruvate. The enzyme from the hyperthermophilic bacterium Thermotoga maritima has been purified and characterized (k_cat ~100 s^?1). It was found that the same enzyme also had the ability to catalyze the production of acetaldehyde and CO₂ from pyruvate, an activity of pyruvate decarboxylase (PDC) at a rate approximately 10% of its AHAS activity. Compared to the catalytic subunit, reconstitution of the individually expressed and purified catalytic and regulatory subunits of the AHAS stimulated both activities of PDC and AHAS. Both activities had similar pH and temperature profiles with an optimal pH of 7.0 and temperature of 85 °C. The enzyme kinetic parameters were determined, however, it showed a non-Michaelis-Menten kinetics for pyruvate only. This is the first report on the PDC activity of an AHAS and the second bifunctional enzyme that might be involved in the production of ethanol from pyruvate in hyperthermophilic microorganisms.     

Cloning and phylogenetic analysis of the genes encoding acetohydroxyacid synthase from the archaeon Methanococcus aeolicus

The gene for acetohydroxyacid synthase (AHAS) was cloned from the archaeon Methanococcus aeolicus. Contrary to biochemical studies [Xing, R. and Whitman, W.B. (1994) J. Bacteriol. 176, 1207–1213] the enzyme was encoded by two open reading frames (ORFs). Based on sequence homology, these ORFs were designated ilvB and ilvN for the large and small subunits of AHAS, respectively. A putative methanogen promoter preceded ilvB-ilvN, and a potential internal promoter was found upstream of ilvN. ilvB encoded a 65-kDa protein, which agreed well with the measured value for the purified enzyme. ilvN encoded a 19-kDa protein, which fell within the range of M_r of small subunits from other sources. Phylogenetic analysis of the deduced amino acid sequence of ilvB showed a close relationship between the AHAS of Bacteria and Archaea, to the exclusion of other enzymes in this family, including pyruvate oxidase, glyoxylate carboligase, pyruvate decarboxylase, and the acetolactate synthase found in fermentative Bacteria. Thus, this family of enzymes probably arose prior to the divergence of the Bacteria and Archaea. Moreover, the higher plant AHAS and the red algal AHAS were related to the AHAS II of Escherichia coli and the cyanobacterial AHAS, respectively. For this reason, these genes appear to have been acquired by the Eucarya during the endosymbiosis that gave rise to the mitochondrion and chloroplast, respectively. One of the ORFs in the Methanococcus jannaschii genome possesses high similarity to the M. aeolicus ilvB, indicating that it is an authentic AHAS.     

Identification of the regulatory subunit of Arabidopsis thaliana acetohydroxyacid synthase and reconstitution with its catalytic subunit

Acetohydroxyacid synthase (EC 4.1.3.18; AHAS) catalyzes the initial step in the formation of the branched-chain amino acids. The enzyme from most bacteria is composed of a catalytic subunit, and a smaller regulatory subunit that is required for full activity and for sensitivity to feedback regulation by valine. A similar arrangement was demonstrated recently for yeast AHAS, and a putative regulatory subunit of tobacco AHAS has also been reported. In this latter case, the enzyme reconstituted from its purified subunits remained insensitive to feedback inhibition, unlike the enzyme extracted from native plant sources. Here we have cloned, expressed in Escherichia coli, and purified the AHAS regulatory subunit of Arabidopsis thaliana. Combining the protein with the purified A. thaliana catalytic subunit results in an activity stimulation that is sensitive to inhibition by valine, leucine, and isoleucine. Moreover, there is a strong synergy between the effects of leucine and valine, which closely mimics the properties of the native enzyme. The regulatory subunit contains a sequence repeat of approximately 180 residues, and we suggest that one repeat binds leucine while the second binds valine or isoleucine. This proposal is supported by reconstitution studies of the individual repeats, which were also cloned, expressed, and purified. The structure and properties of the regulatory subunit are reminiscent of the regulatory domain of threonine deaminase (EC 4.2.1.16), and it is suggested that the two proteins are evolutionarily related.     

Assay of acetohydroxyacid synthase

Acetohydroxyacid synthase (AHAS), also known as acetolactate synthase, has received attention recently because of the finding that it is the site of action of several new herbicides. The most commonly used assay for detecting the enzyme is spectrophotometric involving an indirect detection of the product acetolactate. The assay involves the conversion of the end product acetolactate to acetoin and the detection of acetoin via the formation of a creatine and naphthol complex. There is considerable variability in the literature as to the details of this assay. We have investigated a number of factors involved in detecting AHAS in crude ammonium sulfate precipitates using this spectrophotometric method. Substrate and cofactor saturation levels, pH optimum, and temperature optimum have been determined. We have also optimized a number of factors involved in the generation and the detection of acetoin from acetolactate. The results of these experiments can serve as a reference for new investigators in the study of AHAS.     

The nucleotide sequence of the ilvBN operon of Escherichia coli: sequence homologies of the acetohydroxy acid synthase isozymes.

Three acetohydroxy acid synthase isozymes, AHAS I (ilvBN), AHAS II (ilvGM) and AHAS III (ilvIH) catalyze the first step of the parallel isoleucine-valine biosynthetic pathway in Escherichia coli. Previous DNA sequence and protein purification data have shown that AHAS II and AHAS III are composed of large and small subunits encoded in the ilvGMEDA and ilvIH operons, respectively. Recent protein purification and characterization data have demonstrated that the AHAS I isozyme is also composed of large and small subunits (L. Eoyang, L. and P. M. Silverman [1984] J. Bacteriol. 157:184-189). Now the complete DNA sequence of the operon encoding the AHAS I isozyme has been determined. These data show that both AHAS I subunits (Mr 60,400 and Mr 11,100) are encoded in this operon. The coordinant regulation of both genes of the ilvBN operon has also been demonstrated. Comparisons of the DNA sequences of the genes encoding all three AHAS isozymes have been performed. Conserved homologies were observed between both the large and small subunits of all three isozymes. The closest homology was seen between the AHAS I and AHAS II isozymes. On the basis of these comparisons a rationale for the evolution of the AHAS isozymes in E. coli has been proposed.     

Biosynthesis of Branched-Chain Amino Acids in Schizosaccharomyces pombe: Properties of Acetohydroxy Acid Synthetase1

The regulatory properties of acetohydroxy acid synthetase (AHAS), the first enzyme in the biosynthetic pathway to valine and the second in the isoleucine pathway, were investigated in the fission yeast Schizosaccharomyces pombe. The enzyme was partially purified from crude extracts by protamine sulfate treatment, ammonium sulfate fractionation, and gel filtration through Sephadex G-25. AHAS from S. pombe is unique in that its activity shows a single peak around pH 6.5; high sensitivity to feedback inhibition by valine at this pH (K(i) = 0.1 mM) indicates that the enzyme is involved in valine biosynthesis. Pyruvate saturation kinetics of AHAS extracted from cells grown on glycerol as sole carbon and energy source were normal and hyperbolic. In contrast, the enzyme from glucose-grown cells exhibited sigmoidal saturation kinetics, an effect which disappeared when the synthetase from such cells was partially purified. This phenomenon was shown to be due to competition for pyruvate between AHAS and pyruvate decarboxylase; the latter enzyme is present in large amounts in cells fermenting glucose. Valine inhibition is noncompetitive in nature, and this effector exhibits homotropic cooperative effects; isoleucine is a less-potent inhibitor of AHAS activity. Mercurial treatment reversibly desensitized the enzyme to valine inhibition. On the basis of these data, the S. pombe AHAS appears to be an allosteric regulatory enzyme with the properties of a negative V system.     

Identification of the catalytic subunit of acetohydroxyacid synthase in Haemophilus influenzae and its potent inhibitors

Acetohydroxyacid synthase (AHAS; EC 2.2.1.6) is a thiamin diphosphate- (ThDP)- and FAD-dependent enzyme that catalyzes the first common step in the biosynthetic pathway of the branched-amino acids (BCAAs) leucine, isoleucine, and valine. The gene from Haemophilus influenzae that encodes the AHAS catalytic subunit was cloned, overexpressed in Escherichia coli BL21(DE3), and purified to homogeneity. The purified H. influenzae AHAS catalytic subunit (Hin-AHAS) appeared as a single band on SDS-PAGE gel, with a molecular mass of approximately 63 kDa. The enzyme catalyzes the condensation of two molecules of pyruvate to form acetolactate, with a K(m) of 9.2mM and the specific activity of 1.5 micromol/min/mg. The cofactor activation constant (K(c)=13.5 microM) and the dissociation constant (K(d)=3.3 microM) of ThDP were also determined by enzymatic assay and tryptophan fluorescence quenching studies, respectively. We screened a chemical library to discover new inhibitors of the Hin AHAS catalytic subunit. Through which, AVS-2087 (IC(50)=0.53 microM), KSW30191 (IC(50)=1.42 microM), and KHG20612 (IC(50)=4.91 microM) displayed potent inhibition as compare to sulfometuron methyl (IC(50)=276.31 microM).     

Cloning and characterization of acetohydroxyacid synthase from Bacillus stearothermophilus

Five genes from the ilv-leu operon from Bacillus stearothermophilus have been sequenced. Acetohydroxyacid synthase (AHAS) and its subunits were separately cloned, purified, and characterized. This thermophilic enzyme resembles AHAS III of Escherichia coli, and regulatory subunits of AHAS III complement the catalytic subunit of the AHAS of B. stearothermophilus, suggesting that AHAS III is functionally and evolutionally related to the single AHAS of gram-positive bacteria.     

Characterization of acetohydroxyacid synthase from the hyperthermophilic bacterium Thermotoga maritima

Acetohydroxyacid synthase (AHAS) is the key enzyme in branched chain amino acid biosynthesis pathway. The enzyme activity and properties of a highly thermostable AHAS from the hyperthermophilic bacterium Thermotoga maritima is being reported. The catalytic and regulatory subunits of AHAS from T. maritima were over-expressed in Escherichia coli. The recombinant subunits were purified using a simplified procedure including a heat-treatment step followed by chromatography. A discontinuous colorimetric assay method was optimized and used to determine the kinetic parameters. AHAS activity was determined to be present in several Thermotogales including T. maritima. The catalytic subunit of T. maritima AHAS was purified approximately 30-fold, with an AHAS activity of approximately 160±27 U/mg and native molecular mass of 156±6 kDa. The regulatory subunit was purified to homogeneity and showed no catalytic activity as expected. The optimum pH and temperature for AHAS activity were 7.0 and 85 °C, respectively. The apparent K_m and V_max for pyruvate were 16.4±2 mM and 246±7 U/mg, respectively. Reconstitution of the catalytic and regulatory subunits led to increased AHAS activity. This is the first report on characterization of an isoleucine, leucine, and valine operon (ilv operon) enzyme from a hyperthermophilic microorganism and may contribute to our understanding of the physiological pathways in Thermotogales. The enzyme represents the most active and thermostable AHAS reported so far.     

Determination of products of acetohydroxy acid synthase by the colorimetric method, revisited

The enzyme acetohydroxy acid synthase (AHAS, EC 4.1.3.18) catalyzes two competing reactions of physiological importance: condensation of two molecules of pyruvate to form acetolactate (AL) or condensation of pyruvate and 2-ketobutyrate to form acetohydroxybutyrate (AHB). The activity of AHAS is most frequently analyzed using the Westerfeld method, in which the acetoin formed upon decarboxylation of AL is determined by colorimetric reaction with creatine and alpha-naphthol. However, there has been confusion as to the interpretation of the results of this assay in the presence of both substrates, conditions which lead to formation of both AL and AHB. By applying this assay to enzymatically prepared samples of AL and AHB which have also been analyzed by two other independent methods, we show here that the color yield for AHB in the commonly used assay is 35-40% that for equivalent amounts of acetoin or AL. The relative color yield is not significantly affected by varying the time or temperature of various steps in the color-forming reaction. This information could in principle be used, together with an independent specific assay for AHB, to determine the composition of an AHAS product mixture; it would, however, be less accurate than a simultaneous chromatographic method.