Heterogeneity of autoreactive T cell clones specific for the E2 component of the pyruvate dehydrogenase complex in primary biliary cirrhosis.

The extraordinary specificity of bile duct destruction in primary biliary cirrhosis (PBC) and the presence of T cell infiltrates in the portal tracts have suggested that biliary epithelial cells are the targets of an autoimmune response. The immunodominant antimitochondrial response in patients with PBC is directed against the E2 component of pyruvate dehydrogenase (PDC-E2). Hitherto, there have only been limited reports on the characterization and V beta usage of PDC-E2-specific cloned T cell lines. In this study, we examined peripheral blood mononuclear cells (PBMC) for their reactivity to the entire PDC complex as well as to the E1- and E2-specific components. We also examined the phenotype, lymphokine profile, and V beta usage of PDC-specific T cell clones isolated from cellular infiltrates from the livers of PBC patients. We report that PBMC from 16/19 patients with PBC, but not 12 control patients, respond to the PDC-E2 subunit. Interestingly, this response was directed to the inner and/or the outer lipoyl domains, despite the serologic observation that the autoantibody response is directed predominantly to the inner lipoyl domain. Additionally, lymphokine analysis of interleukin (IL) 2/IL-4/interferon gamma production from individual liver-derived autoantigen-specific T cell clones suggests that both T helper cell Th1- and Th2-like clones are present in the liver. Moreover, there was considerable heterogeneity in the T cell receptor for antigen (TCR) V beta usage of these antigen-specific autoreactive T cell clones. This is in contrast to murine studies in which animals are induced to develop autoimmunity by specific immunization and have an extremely limited T cell V beta repertoire. Thus, our data suggest that in human organ-specific autoimmune diseases, such as PBC, the TCR V beta repertoire is heterogenous.

Identification and precursor frequency analysis of a common T cell epitope motif in mitochondrial autoantigens in primary biliary cirrhosis.

The immunodominant antimitochondrial antibody response in patients with primary biliary cirrhosis (PBC) is directed against the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Based on our earlier observations regarding peripheral blood mononuclear cell (PBMC) T cell epitopes, we reasoned that a comparative analysis of the precursor frequencies of PDC-E2 163-176-specific T cells isolated from PBMC, regional hepatic lymph nodes, and from the liver of PBC patients would provide insight regarding the role of T cells in PBC. Results showed a disease-specific 100-150-fold increase in the precursor frequency of PDC-E2 163-176-specific T cells in the hilar lymph nodes and liver when compared with PBMC from PBC patients. Interestingly, autoreactive T cells and autoantibodies from PBC patients both recognize the same dominant epitope. In addition, we demonstrated cross-reactivity of PDC-E2 peptide 163-176-specific T cell clones with PDC-E2 peptide 36-49 and OGDC-E2 peptide 100-113 thereby identifying a common T cell epitope "motif" ExETDK. The peptide 163-176-specific T cell clones also reacted with purified native PDC-E2, suggesting that this epitope is not a cryptic determinant. These data provide evidence for a major role for PDC-E2 peptide 163-176 and/or peptides bearing a similar motif in the pathogenesis of PBC.

Distinct regulatory properties of pyruvate dehydrogenase kinase and phosphatase isoforms.

The mammalian pyruvate dehydrogenase complex (PDC) plays central and strategic roles in the control of the use of glucose-linked substrates as sources of oxidative energy or as precursors in the biosynthesis of fatty acids. The activity of this mitochondrial complex is regulated by the continuous operation of competing pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP) reactions. The resulting interconversion cycle determines the fraction of active (nonphosphorylated) pyruvate dehydrogenase (E1) component. Tissue-specific and metabolic state-specific control is achieved by the selective expression and distinct regulatory properties of at least four PDK isozymes and two PDP isozymes. The PDK isoforms are members of a family of serine kinases that are not structurally related to cytoplasmic Ser/Thr/Tyr kinases. The catalytic subunits of the PDP isoforms are Mg2+-dependent members of the phosphatase 2C family that has binuclear metal-binding sites within the active site. The dihydrolipoyl acetyltransferase (E2) and the dihydrolipoyl dehydrogenase-binding protein (E3BP) are multidomain proteins that form the oligomeric core of the complex. One or more of their three lipoyl domains (two in E2) selectively bind each PDK and PDP1. These adaptive interactions predominantly influence the catalytic efficiencies and effector control of these regulatory enzymes. When fatty acids are the preferred source of acetyl-CoA and NADH, feedback inactivation of PDC is accomplished by the activity of certain kinase isoforms being stimulated upon preferentially binding a lipoyl domain containing a reductively acetylated lipoyl group. PDC activity is increased in Ca2+-sensitive tissues by elevating PDP1 activity via the Ca2+-dependent binding of PDP1 to a lipoyl domain of E2. During starvation, the irrecoverable loss of glucose carbons is restricted by minimizing PDC activity due to high kinase activity that results from the overexpression of specific kinase isoforms. Overexpression of the same PDK isoforms deleteriously hinders glucose consumption in unregulated diabetes.

Arginine-239 in the beta subunit is at or near the active site of bovine pyruvate dehydrogenase.

We have modified bovine pyruvate dehydrogenase (E1), the first catalytic component of the pyruvate dehydrogenase complex, with pyreneglyoxal. Treatment of E1 with pyreneglyoxal resulted in the loss of enzyme activity. Pyruvate plus thiamin pyrophosphate (TPP) afforded approximately 80% protection against this inactivation and protected two arginine residues per mol of E1 tetramer (alpha 2 beta 2) from modification. Circular dichroism spectral analysis indicated absence of any gross structural changes in the enzyme as a result of modification. Comparison of the peptide maps, monitored at 345 nm of unprotected and pyruvate plus TPP protected E1s after V8 digestion revealed that a peptide in the protected enzyme was labeled by pyreneglyoxal to a lesser extent than its counterpart in the unprotected enzyme. Sequence analysis of the peptide demonstrated that it corresponded precisely to amino-acid residues 235 to 246 in the human E1 beta sequence, with arginine residues at positions 239 and 242. Since Arg-239 is conserved in the beta-subunit of all presently known sequences of the pyruvate dehydrogenase complex and branched-chain alpha-keto acid dehydrogenase complex, it is strongly suggested that Arg-239 in the human E1 beta sequence is at or near the active site of bovine E1.

Sepharose-insolubilization of the dihydrolipoyl transacetylase core component of the pyruvate dehydrogenase complex: preparation and characterization.

The dihydrolipoyl transacetylase core components of the bovine kidney and heart pyruvate dehydrogenase complexes were covalently attached through the lipoyl moiety to Sepharose by the thiol-crosslinking reagent, N,N'-p-phenylenedimaleimide. In one approach, the N,N-p-phenylenedimaleimide was allowed to react with glutathione which was in turn linked by its N-terminal to Sepharose CL-6B. In addition, we found that N,N-p-phenylenedimaleimide would react directly with Sepharose CL-6B (at undetermined sites) and could be used as the sole bridge in forming a stable linkage of the transacetylase core to Sepharose. With the latter approach the extent of multiple-linkage of the 60-subunit core could more easily be controlled. This should be a generally useful approach for linking proteins with reactive surface thiol residues. Insolubilization of the core of the pyruvate dehydrogenase complex by these methods did not appear to significantly alter the binding of other protein components of the complex, but the catalytic activities of the complex requiring the lipoyl moiety were appreciably altered. Procedures for coupling the transacetylase core to various derivatives of phenylenedimaleimide-Sepharose and techniques described for studying the protein products should be useful in preparation of specialized matrices for both protein purification and the study of protein-protein interactions.

Pyruvate dehydrogenase kinase regulatory mechanisms and inhibition in treating diabetes, heart ischemia, and cancer.

The fraction of pyruvate dehydrogenase complex (PDC) in the active form is reduced by the activities of dedicated PD kinase isozymes (PDK1, PDK2, PDK3 and PDK4). Via binding to the inner lipoyl domain (L2) of the dihydrolipoyl acetyltransferase (E2 60mer), PDK rapidly access their E2-bound PD substrate. The E2-enhanced activity of the widely distributed PDK2 is limited by dissociation of ADP from its C-terminal catalytic domain, and this is further slowed by pyruvate binding to the N-terminal regulatory (R) domain. Via the reverse of the PDC reaction, NADH and acetyl-CoA reductively acetylate lipoyl group of L2, which binds to the R domain and stimulates PDK2 activity by speeding up ADP dissociation. Activation of PDC by synthetic PDK inhibitors binding at the pyruvate or lipoyl binding sites decreased damage during heart ischemia and lowered blood glucose in insulin-resistant animals. PDC activation also triggers apoptosis in cancer cells that selectively convert glucose to lactate.

Inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex by reduced nicotinamide adenine dinucleotide in the presence or absence of calcium ion and effect of adenosine 5'-diphosphate on reduced nicotinamide adenine dinucleotide inhibition.

Micromolar Ca2+ markedly reduces NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex [Lawlis, V. B., & Roche, T. E. (1980) Mol. Cell. Biochem. 32, 147-152]. Product inhibition patterns from initial velocity studies conducted at less than 10(-9) M or at 1.5 X 10(-5) M Ca2+ with NAD+, CoA, or alpha-ketoglutarate as the variable substrate showed that NADH was a noncompetitive inhibitor with respect to each of these substrates, except at high NAD+ concentrations, where reciprocal plots were nonlinear and the inhibition pattern for NADH vs. NAD+ changed from a noncompetitive to a competitive pattern. From slope and intercept replots, 2-fold to 12-fold higher inhibition constants were estimated for inhibition by NADH vs. the various substrates in the presence of 1.5 X 10(-5) M Ca2+ than for inhibition at less than 10(-9) M Ca2+. These inhibition patterns and the lack of an effect of Ca2+ on the inhibition of the dihydrolipoyl dehydrogenase component suggested that Ca2+-modulated NADH inhibition occurs at an allosteric site with competitive binding at the site by high levels of NAD+. Decarboxylation of alpha-keto[1-14C]glutarate by the resolved alpha-ketoglutarate dehydrogenase component was investigated in the presence of 5.0 mM glyoxylate which served as an efficient acceptor. NADH (0.2 mM) or 1.0 mM ATP inhibited the partial reaction whereas 15 muM Ca2+, 1.0 mM ADP, or 10 mM NAD+ stimulated the partial reaction and reduced NADH inhibition of this reaction. Thus these effectors alter the activity of the alpha-ketoglutarate dehydrogenase complex by binding at allosteric sites on the alpha-ketoglutarate dehydrogenase component. Inhibition by NADH over a wide range of NADH/NAD+ ratios was measured under conditions in which the level of alpha-ketoglutarate was adjusted to give matching control activities at less than 10(-9) M Ca2+ or 1.5 X 10(-5) M Ca2+ in either the presence or the absence of 1.6 mM ADP. These studies establish that both Ca2+ and ADP decreased NADH inhibition under conditions compensating for the effects of Ca2+ and ADP on S0.5 for alpha-ketoglutarate. ADP was particularly effective in reducing NADH inhibition; further studies are required to determine whether this occurs through binding of NADH and ADP at the same, overlapping, or interacting sites.

Effect of micromolar Ca2+ on NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex and possible role of Ca2+ in signal amplification.

NADH inhibition of bovine kidney alpha-ketoglutarate dehydrogenase complex was compared at 10 microM free Ca2+ or in the absence of Ca2+ (i.e., less than 1.0 nM free Ca2+). In the presence of Ca2+, NADH inhibition was appreciably decreased for a wide range of NADH:NAD+ ratios. A half-maximal decrease in NADH inhibition occurred at slightly less than 1 microM free Ca/+ (as determined with EGTA-Ca buffers). Of necessity this was observed on top of an effect of Ca2+ on the S0.5 for alpha-ketoglutarate which was decreased by Ca2+ with a half-maximal effect at a similar concentration. The effect of Ca2+ on NADH inhibition was not observed in assays of the dihydrolipoyl dehydrogenase component (using dihydrolipoamide as a substrate) or in assays of bovine kidney pyruvate dehydrogenase complex. This indicates that the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex is required to elicit the effect of Ca2+ on NADH inhibition. At a fixed alpha-ketoglutarate concentration (50 microM), removal of Ca2+ reduced the activity of the alpha-ketoglutarate dehydrogenase complex by 8.5-fold (due to an increase in S0.5 for alpha-ketoglutarate) and, in the presence of different NADH:NAD+ ratios, decreased the activity of the complex by 50 to 100-fold. Effects of the phosphate potential (ATP/ADPxPi) or a combination of the phosphate potential and NADH:NAD+ ratio are also described. The possibility that the level of intramitochondrial free Ca/+ serves as a signal amplifier normally coupled to the energy state of mitochondria is discussed.

Modification of bovine kidney pyruvate dehydrogenase kinase activity by CoA esters and their mechanism of action.

The activation of pyruvate dehydrogenasea kinase activity by CoA esters has been further characterized. Half-maximal activation of kinase activity was achieved with about 1.0 microM acetyl-CoA after a 20-s preincubation in the presence of NADH. More than 80% of the acetyl-CoA was consumed during this period in acetylating sites in the pyruvate dehydrogenase complex as a result of the transacetylation reaction proceeding to equilibrium. At 1.0 microM acetyl-CoA, this resulted in more than a 4-fold higher level of CoA than residual acetyl-CoA. Activation of kinase activity could result either from acetylation of specific sites in the complex or tight binding of acetyl-CoA. Removal of CoA enhanced both acetylation and activation, suggesting acetylation mediates activation. For allosteric binding of acetyl-CoA to elicit activation, an activation constant, Ka, less than 50 nM would be required. To further distinguish between those mechanisms, the effects of other CoA esters as well as the reactivity of most of the effective CoA esters were characterized. Several short-chain CoA esters enhanced kinase activity including (in decreasing order of effectiveness) malonyl-CoA, acetoacetyl-CoA, propionyl-CoA, and methylmalonyl-CoA. Butyryl-CoA inhibited kinase activity as did high concentrations of long-chain acyl-CoAs. Inhibition by long-chain acyl-CoAs may result, in part, from detergent-like properties of those esters. Malonyl-CoA, propionyl-CoA, butyryl-CoA, and methylmalonyl-CoA, obtained with radiolabeled acyl groups, were shown to acylate sites in the complex. Propionyl-CoA and butyryl-CoA were tested, in competition with acetyl-CoA or pyruvate, as alternative substrates for acylation of sites in the complex and as competitive effectors of kinase activity. Propionyl-CoA alone rapidly acylated sites in the complex at low concentrations, and low concentrations of propionyl-CoA were effective in activating kinase activity although only a relatively small activation was observed. When an equivalent level (20 microM) of acetyl-CoA and propionyl-CoA was used, marked activation of kinase activity due to a dominant effect of acetyl-CoA was associated with acetylation of a major portion of sites in the complex and with a small portion undergoing acylation with propionyl-CoA. Those results were rapidly achieved in a manner independent of the order of addition of the two CoA esters. That indicates that tight slowly reversible binding of acetyl-CoA is not involved in kinase activation. High levels of propionyl-CoA greatly reduced acetylation by acetyl-CoA and nearly prevented activation of kinase activity by acetyl-CoA.(ABSTRACT TRUNCATED AT 400 WORDS)

Regulation of bovine kidney alpha-ketoglutarate dehydrogenase complex by calcium ion and adenine nucleotides. Effects on S0.5 for alpha-ketoglutarate.

Regulation of bovine kidney alpha-ketoglutarate dehydrogenase complex by energy-linked metabolites was investigated. Ca2+, ADP, or inorganic phosphate markedly enhanced the activity of the complex, and ATP or, to a lesser extent, GTP decreased the activity of the complex. Initial velocity studies with alpha-ketoglutarate as the varied substrate demonstrated that these modulators induced large changes in S0.5 for alpha-ketoglutarate (based on analysis in Hill plots) with no change in the maximum velocity (as determined by double-reciprocal plots). For all conditions studied, the Hill coefficients were significantly less than 1.0 with slopes that were linear over wide ranges of alpha-ketoglutarate concentrations, indicating negative cooperativity that probably resulted from multiple site-site interactions. Ca2+ (maintained at 10 muM by a Ca2+ buffer) decreased the S0.5 for alpha-ketoglutarate 63-fold (from 25 to 0.40 mM); even in the presence of a positive effector, ADP or phosphate, Ca2+ decreased the S0.5 for alpha-ketoglutarate 7.8- or 28-fold, respectively. Consistent with a mechanism of action dependent of Ca2+, ADP (1.60 mM) or phosphate (20 mM) reduced the S0.5 for alpha-ketoglutarate in the presence of Ca2+ (i.e., 4.5- or 1.67-fold, respectively); however, these effectors elicited larger decreases in S0.5 in the absence of Ca2+ (i.e., 37- or 3.7-fold, respectively). ATP (1.6 mM) increased the S0.5 for alpha-ketoglutarate, and Ca2+ appreciably reduced the effect, lowering the S0.5 98-fold from 66 to 0.67 mM. Thus the activity of the kidney alpha-ketoglutarate dehydrogenase complex is poised to increase as the energy potential in mitochondria declines, and Ca2+ has a pronounced modulatory effect. Comparative studies on bovine heart alpha-ketoglutarate dehydrogenase complex and the effects of varying the ADP/ATP ratio in the presence or absence of Ca2+ or phosphate are also described.

Component requirements for NADH inhibition and spermine stimulation of pyruvate dehydrogenaseb phosphatase activity.

The subunit and subdomain requirements for NADH inhibition as well as Ca+ and spermine activation of the pyruvate dehydrogenaseb phosphatase were analyzed. The transacetylase-protein X subcomplex (E2-X) was required for all three effects. The oligomeric inner domain of the transacetylase did not support any of these regulatory effects. The presence of at least a portion of the outer (lipoyl-bearing) domains of the transacetylase but not the lipoyl-bearing portion of protein X was essential for expression of these regulatory effects on phosphatase activity. The inner domain of protein X may contribute to some effects. The E2-X subcomplex, alone, had no effect on phosphatase activity in the absence of Ca2+, but the subcomplex did support both NADH inhibition and spermine activation in the absence of Ca2+. Studies with peptide substrates established that spermine is directly bound by a phosphatase subunit. With the resolved pyruvate dehydrogenase component (E1b) used as the substrate, the E2-X subcomplex transformed the effect of spermine from inhibiting to stimulating the rate of dephosphorylation by the phosphatase. The above observations suggest that binding of E1b to the E2-X subcomplex alters its presentation to the phosphatase. We also present several observations that are consistent with NADH inhibition of the phosphatase being mediated through a dihydrolipoyl dehydrogenase-dependent reduction of lipoyl moieties in the E2-X subcomplex. Overall, our data establish that the outer, lipoyl-bearing domains of the oligomeric transacetylase core have an essential role in the function and regulation of the pyruvate dehydrogenase phosphatase.

The catalytic requirements for reduction and acetylation of protein X and the related regulation of various forms of resolved pyruvate dehydrogenase kinase.

The pyruvate dehydrogenase kinase consists of a catalytic subunit (Kc) and a basic subunit (Kb) which appear to be anchored to the dihydrolipoyl transacetylase core component (E2) by another subunit, referred to as protein X (Rahmatullah, M., Jilka, J. M., Radke, G. A., and Roche, T. E. (1986) J. Biol. Chem. 261, 6515-6523). We determined the catalytic requirements for reduction and acetylation of the lipoyl moiety in protein X and linked those changes in protein X to regulatory effects on kinase activity. Using fractions prepared by resolution and proteolytic treatments, we evaluated which subunits are required for regulatory effects on kinase activity. With X-KcKb fraction (treated to remove the mercurial agent used in its preparation), we found that the resolved pyruvate dehydrogenase component, the isolated inner domain of E2 (lacking the lipoyl-bearing region of E2), and the dihydrolipoyl dehydrogenase component directly utilize protein X as a substrate. The resulting reduction and acetylation of protein X occurs in association with enhancement of kinase activity. Following tryptic cleavage of E2 and protein X into subdomains, full acetylation of the lipoyl-bearing subdomains of these proteins is retained along with the capacity of acetylating substrates to stimulate kinase activity. All kinase-containing fractions, including those in which the Kb subunit was digested, were inhibited by pyruvate or ADP, alone, and synergistically by the combination suggesting that pyruvate and ADP bind to Kc. Our results suggest that the Kb subunit of the kinase does not contribute to the observed regulatory effects. A dynamic role of protein X in attenuating kinase activity based on changes in the mitochondrial redox and acetylating potentials is considered.

Molecular biology and biochemistry of pyruvate dehydrogenase complexes.

In most organisms, the pyruvate dehydrogenase complex catalyzes the pivotal irreversible reaction that leads to the consumption of glucose in the aerobic, energy-generating pathways. A combination of biochemical and molecular biology studies have greatly expanded our understanding of the overall structural organization of this multicomponent system, delineated the locations and elucidated the functions of structural domains of the catalytic components, and revealed significant evolutionary changes. Important to this progress was the deduction of the primary amino acid sequences from cDNA clones for each of the catalytic components from several species. The greatest detail is available for the FAD-containing dihydrolipoamide dehydrogenase component, which is the only component for which tertiary structure information has recently emerged. For the dihydrolipoamide acetyltransferase core component, a similar but species-variable multidomain structure is established that is responsible for the distinct architectures of the inner cores, the peripheral binding of the other components, and the conveyance of reaction intermediates between distantly separated active sites. A second lipoyl-bearing component, protein X, has been shown to play a critical role in the organization and function of the complex from many higher organisms. Although much is known about the means of effector modulation of mammalian complex activity, identification of the signal eliciting its regulation by insulin still poses an exciting challenge.

Spectrophotometric assay of the flavin-containing monooxygenase and changes in its activity in female mouse liver with nutritional and diurnal conditions.

A highly sensitive spectrophotometric assay was developed for measuring flavin-containing monooxygenase activity using methimazole (N-methyl-2-mercaptoimidazole) as the substrate. With the procedure described, flavin-containing monooxygenase activity can be accurately measured in whole cell homogenates without interference due to NADPH oxidase activities. The effects of detergents and octylamine on female mouse liver flavin-containing monooxygenase activity were characterized for whole homogenates and microsomes prepared under conditions which tend to cause or minimize microsomal aggregation. A small activation was observed with 0.2% (v/v) Emulgen 913 with nonaggregated microsomes; higher levels of detergents gave maximal activity with aggregated microsomes. Variations in the activity of the female mouse liver enzyme with nutritional state and time of day were evaluated. Higher specific activities were observed in homogenates and microsomes of livers from fed animals than from livers of 24-h starved animals, and higher specific activities were present in samples from livers of animals sacrificed in late afternoon than in the early morning. In the period where activity increased in fed animals (i.e., the AM to PM transition), a portion of flavin-containing monooxygenase was more resistant to thermal inactivation. Other properties are described which suggest structural differences for at least a portion of the flavin-containing monooxygenase. The possibility that these differences may be related to turnover of the flavin-containing monooxygenase is discussed.

Specificity of the pyruvate dehydrogenase kinase for pyruvate dehydrogenase component bound to the surface of the kidney pyruvate dehydrogenase complex and evidence for intracore migration of pyruvate dehydrogenase component.

Using the bovine kidney pyruvate dehydrogenase complex we have investigated the mechanism whereby about three pyruvate dehydrogenase (active form) kinase molecules, tightly bound to the dihydrolipoyl transacetylase core, can rapidly phosphorylate and inactivate about 20 pyruvate dehydrogenase (active form) (PDHa) tetramers which are also bound to the 60-subunit core. Evidence is presented that PDHa kinase activity is not serviced by a process of dissociation and reassociation of PDHa. Rapid inactivation of a full complement of PDHa occurs at a rate exceeding the rate of dissociation of PDHa, indicating that a PDHa must move to the fixed kinase subunits without dissociating from the dihydrolipoyl transacetylase core. Consistent with that concept, at low concentrations of complex where a significant portion of PDHa is free, bound PDHa was inactivated at a rate equivalent to that at higher concentrations of complex, and free PDHa was phosphorylated more slowly at a rate closely approximated by the rate of association of free PDHa with the transacetylase core. Thus, with a low number of PDHa molecules bound, PDHa either is preferentially positioned for phosphorylation and inactivation by PDHa kinase or can rapidly become so positioned without dissociating from the transacetylase core.

Identification of the essential cysteine residue in the active site of bovine pyruvate dehydrogenase.

Pyruvate dehydrogenase (E1), the first catalytic component of the bovine pyruvate dehydrogenase complex, is composed of two nonidentical subunits in a tetrameric alpha 2 beta 2 form. The sulfhydryl-specific reagent N-ethylmaleimide (NEM) was used to identify the reactivities and function of cysteinyl residues and subsequent identification of these residues in the active site of bovine E1. Treatment of E1 with 0.2 mM NEM resulted in loss (90%) of enzymatic activity; the inactivation followed bimolecular reaction kinetics. The inactivation was almost entirely prevented by thiamin pyrophosphate (TPP) and pyruvate; protection is probably due to formation of the hydroxyethylidene-TPP intermediate. To identify the reactive cysteinyl residues in the active site region, the nonessential SH groups in E1 were first modified with NEM in the presence of TPP and pyruvate. After quenching with dithiothreitol and removal of the substrate and cofactor by dialysis, the modified E1 was treated with [14C]NEM to label the exposed cysteinyl residue(s) in or near the active site region. The data indicate that NEM reacted in the active site region of the E1 component with a stoichiometry of 2 mol of [14C]NEM bound per mol of E1 tetramer. The initial rapid labeling of E1 with [14C]NEM established that incorporation was predominantly into the alpha subunit. A single radiolabeled peptide was isolated following V8 protease digestion of radiolabeled E1 by [14C]NEM. Sequence analysis of the labeled peptide derived from bovine E1 demonstrated that the labeled cysteinyl residue was equivalent to Cys-62 in the alpha subunit (mature form) of human E1.