Overall kinetic mechanism of saccharopine dehydrogenase from Saccharomyces cerevisiae

Kinetic data have been measured for the histidine-tagged saccharopine dehydrogenase from Saccharomyces cerevisiae, suggesting the ordered addition of nicotinamide adenine dinucleotide (NAD) followed by saccharopine in the physiologic reaction direction. In the opposite direction, the reduced nicotinamide adenine dinucleotide (NADH) adds to the enzyme first, while there is no preference for the order of binding of alpha-ketoglutarate (alpha-Kg) and lysine. In the direction of saccharopine formation, data also suggest that, at high concentrations, lysine inhibits the reaction by binding to free enzyme. In addition, uncompetitive substrate inhibition by alpha-Kg and double inhibition by NAD and alpha-Kg suggest the existence of an abortive E:NAD:alpha-Kg complex. Product inhibition by saccharopine is uncompetitive versus NADH, suggesting a practical irreversibility of the reaction at pH 7.0 in agreement with the overall K(eq). Saccharopine is noncompetitive versus lysine or alpha-Kg, suggesting the existence of both E:NADH:saccharopine and E:NAD:saccharopine complexes. NAD is competitive versus NADH, and noncompetitive versus lysine and alpha-Kg, indicating the combination of the dinucleotides with free enzyme. Dead-end inhibition studies are also consistent with the random addition of alpha-Kg and lysine. Leucine and oxalylglycine serve as lysine and alpha-Kg dead-end analogues, respectively, and are uncompetitive against NADH and noncompetitive against alpha-Kg and lysine, respectively. Oxaloacetate (OAA), pyruvate, and glutarate behave as dead-end analogues of lysine, which suggests that the lysine-binding site has a higher affinity for keto acid analogues than does the alpha-Kg site or that dicarboxylic acids have more than one binding mode on the enzyme. In addition, OAA and glutarate also bind to free enzyme as does lysine at high concentrations. Glutarate gives S-parabolic noncompetitive inhibition versus NADH, indicating the formation of a E:(glutarate)2 complex as a result of occupying both the lysine- and alpha-Kg-binding sites. Pyruvate, a slow alternative keto acid substrate, exhibits competitive inhibition versus both lysine and alpha-Kg, suggesting the combination to the E:NADH:alpha-Kg and E:NADH:lysine enzyme forms. The equilibrium constant for the reaction has been measured at pH 7.0 as 3.9 x 10(-7) M by monitoring the change in NADH upon the addition of the enzyme. The Haldane relationship is in very good agreement with the directly measured value.     

Overall kinetic mechanism of saccharopine dehydrogenase (L-glutamate forming) from Saccharomyces cerevisiae

Kinetic studies were carried out for histidine-tagged saccharopine reductase from Saccharomyces cerevisiae at pH 7.0, suggesting a sequential mechanism with ordered addition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) to the free enzyme followed by L-alpha-aminoadipate-delta-semialdehyde ( L-AASA) which adds in rapid equilibrium prior to l-glutamate in the forward reaction direction. In the reverse reaction direction, nicotinamide adenine dinucleotide phosphate (NADP) adds to the enzyme followed by addition of saccharopine. Product inhibition by NADP is competitive vs NADPH and noncompetitive vs alpha-AASA and L-glutamate, suggesting that the dinucleotide adds to the free enzyme prior to the aldehyde. Saccharopine is noncompetitive vs NADPH, alpha-AASA, and L-glutamate. In the direction of saccharopine oxidation, NADPH is competitive vs NADP and noncompetitive vs saccharopine, L-glutamate is noncompetitive vs both NADP and saccharopine, while L-AASA is noncompetitive vs saccharopine and uncompetitive vs NADP. The sequential mechanism is also corroborated by dead-end inhibition studies using analogues of AASA, L-glutamate, and saccharopine. 2-Amino-6-heptenoic acid was chosen as a dead-end analogue of L-AASA and is competitive vs AASA, uncompetitive vs NADPH, and noncompetitive vs L-glutamate. alpha-Ketoglutarate (alpha-Kg) serves as the dead-end analogue of L-glutamate and is competitive vs L-glutamate and uncompetitive vs L-AASA and NADPH. In the direction of saccharopine oxidation, N-oxalylglycine, L-pipecolic acid, L-leucine, alpha-ketoglutarate, glyoxylic acid, and L-ornithine were used as dead-end analogues of saccharopine and showed competitive inhibition vs saccharopine and uncompetitive inhibition vs NADP. The equilibrium constant for the reaction was measured at pH 7.0 by monitoring the change in absorbance of NADPH and is 200 M(-1). The value is in good agreement with the value determined using the Haldane relationship.     

Mycophenolic acid and thiazole adenine dinucleotide inhibition of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase: implications on enzyme mechanism

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the oxidation of inosine 5'-monophosphate (IMP) to xanthosine 5'-monophosphate (XMP) with the conversion of NAD to NADH. An ordered sequential mechanism where IMP is the first substrate bound and XMP is the last product released was proposed for Tritrichomonas foetus IMPDH on the basis of product inhibition studies. Thiazole adenine dinucleotide (TAD) is an uncompetitive inhibitor versus IMP and a noncompetitive inhibitor versus NAD, which suggests that TAD binds to both E-IMP and E-XMP. Mycophenolic acid is also an uncompetitive inhibitor versus IMP and noncompetitive versus NAD. Multiple-inhibitor experiments show that TAD and mycophenolic acid are mutually exclusive with each other and with NADH. Therefore, mycophenolic acid most probably binds to the dinucleotide site of T. foetus IMPDH. The mycophenolic acid binding site was further localized to the nicotinamide subsite within the dinucleotide site: mycophenolic acid was mutually exclusive with tiazofurin, but could form ternary enzyme complexes with ADP or adenosine diphosphate ribose. NAD inhibits the IMPDH reaction at concentrations greater than 3 mM. NAD substrate inhibition is uncompetitive versus IMP, which suggests that NAD inhibits by binding to E-XMP. TAD is mutually exclusive with both NAD and NADH in multiple-inhibitor experiments, which suggests that there is one dinucleotide binding site. The ordered mechanism predicts that multiple-inhibitor experiments with XMP and TAD, mycophenolic acid, or NAD should have an interaction constant (alpha) between 0 and 1. However, alpha was greater than 1 in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic analysis of duck epsilon-crystallin, a lens structural protein with lactate dehydrogenase activity.

Biochemical characterization and kinetic analysis of epsilon-crystallin from the lenses of common ducks were undertaken to elucidate the enzyme mechanism of this unique crystallin with lactate dehydrogenase (LDH) activity. Despite the structural similarities between epsilon-crystallin and chicken heart LDH, differences in charge and kinetic properties were revealed by isoenzyme electrophoresis and kinetic studies. Bi-substrate kinetic analysis examined by initial-velocity and product-inhibition studies suggested a compulsory ordered Bi Bi sequential mechanism with NADH as the leading substrate followed by pyruvate. The products were released in the order L-lactate and NAD+. The catalysed reaction is shown to have a higher rate in the formation of L-lactate and NAD+. Substrate inhibition was observed at high concentrations of pyruvate and L-lactate for the forward and reverse reactions respectively. The substrate inhibition was presumably due to the formation of epsilon-crystallin-NAD(+)-pyruvate or epsilon-crystallin-NADH-L-lactate abortive ternary complexes, as suggested by the product-inhibition studies. The significance and the interrelationship of duck epsilon-crystallin with other well-known LDHs are discussed with special regard to its role as a structural protein with some enzymic function in lens metabolism.     

Ternary complex formation of pig heart lactate dehydrogenase with spin-labelled coenzyme and inhibitors as studied by electron spin resonance.

The formation of ternary inhibitor and 'dead end' complexes of pig heart lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) was studied by means of two NAD derivatives, spin-labelled at N6 and C-8 of the adenine ring. Dissociation constants calculated for the inhibitors oxamate and oxalate from their corresponding ternary complexes are in excellent agreement with data from literature derived from sedimentation experiments. However, the recently postulated enzyme-NADH-sulfite complex was not observed. The mobility of the spin-label, i.e. the protein conformation near the adenine binding pocket in various ternary complexes depends on the type of inhibition or substrate employed.     

Role of ions in the regulation of porcine lactate dehydrogenase.

Different ions affect the H4 and M4 isoenzymes of porcine lactate dehydrogenase (L-lactate: NAD+ oxidoreductase, EC 1.1.1.27) in the same way, inhibiting the enzyme at low pyruvate concentrations, whereas at high pyruvate concentrations, the activities were enhanced. The inhibition was competitive with regard to pyruvate and NADH. The enhancement of the enzyme activity at high pyruvate concentration is due to the increase in the Km value for pyruvate, implying that higher substrate concentrations are needed to obtain substrate inhibition. Sulphate behaved differently from the other ions. It inhibited in a noncompetitive manner with regard to pyruvate and did not activate the enzyme at high pryvuate concentration. The effect of ions increased with the size of the anion. The ionic strength was of less importance.     

Rate-limiting steps for hepatic gluconeogenesis. Mechanism of oxamate inhibition of mitochondrial pyruvate metabolism

Oxamate, structural analog of pyruvate, inhibits gluconeogenesis from pyruvate or substrates yielding pyruvate. The inhibitory effect is the result of a decreased mitochondrial pyruvate utilization. Although the inhibition of gluconeogenesis is competitive for pyruvate, in isolated mitochondria oxamate displays a mixed type kinetics inhibitory pattern of pyruvate utilization. Evidence is presented indicating that this mixed type pattern of inhibition is the result of the action of oxamate on two different sites: noncompetitive inhibition of pyruvate carboxylation, and competitive inhibition of pyruvate entry into the mitochondria. At concentrations of pyruvate above 0.4 mM, although pyruvate carboxylation is decreased by 40% by oxamate, no detectable effects on the gluconeogenic flux were observed. This finding strongly indicates that pyruvate carboxylase is not an important rate-limiting step for hepatic gluconeogenesis. Thus, the inhibition of gluconeogenesis at low pyruvate concentrations (less than 0.4 mM) seems to be the result of an interaction of oxamate with the mitochondrial pyruvate translocator, indicating that pyruvate transport across the mitochondrial membrane is the first nonequilibrium step in the gluconeogenic pathway when low physiological concentrations of this substrate are utilized.     

Probing Aplysia californica adenosine 5'-diphosphate ribosyl cyclase for substrate binding requirements: design of potent inhibitors.

Readily synthesized nicotinamide adenine dinucleotide (NAD(+)) analogues have been used to investigate aspects of the cyclization of NAD(+) to cyclic adenosine 5'-O-diphosphate ribose (cADPR) catalyzed by the enzyme adenosine 5'-O-diphosphate (ADP) ribosyl cyclase and to produce the first potent inhibitors of this enzyme. In all cases, inhibition of Aplysia californica cyclase by various substrate analogues was found to be competitive while inhibition by nicotinamide exhibited mixed-behavior characteristics. Nicotinamide hypoxanthine dinucleotide (NHD(+)), nicotinamide guanine dinucleotide (NGD(+)), C1'-m-benzamide adenine dinucleotide (Bp(2)A), and C1'-m-benzamide nicotinamide dinucleotide (Bp(2)N) were found to be nanomolar potency inhibitors with inhibition constants of 70, 143, 189, and 201 nM, respectively. However, NHD(+) and NGD(+) are also known substrates and are slowly converted to cyclic products, thus preventing their further use as inhibitors. The symmetrical bis-nucleotides, bis-adenine dinucleotide (Ap(2)A), bis-hypoxanthine dinucleotide (Hp(2)H), and bis-nicotinamide dinucleotide (Np(2)N), exhibited micromolar competitive inhibition, with Ap(2)A displaying the greatest affinity for the enzyme. 2',3'-Di-O-acetyl nicotinamide adenine dinucleotide (AcONAD(+)) was not a substrate for the A. californica cyclase but also displayed some inhibition at a micromolar level. Finally, inhibition of the cyclase by adenosine 5'-O-diphosphate ribose (ADPR) and inosine 5'-O-diphosphate ribose (IDPR) was observed at millimolar concentration. The nicotinamide aromatic ring appears to be the optimal motif required for enzymatic recognition, while modifications of the 2'- and 3'-hydroxyls of the nicotinamide ribose seem to hamper binding to the enzyme. Stabilizing enzyme/inhibitor interactions and the inability of the enzyme to release unprocessed material are both considered to explain nanomolar inhibition. Recognition of inhibitors by other ADP ribosyl cyclases has also been investigated, and this study now provides the first potent nonhydrolyzable sea urchin ADP ribosyl cyclase and cADPR hydrolase inhibitor Bp(2)A, with inhibition observed at the micromolar and nanomolar level, respectively. The benzamide derivatives did not inhibit CD38 cyclase or hydrolase activity when NGD(+) was used as substrate. These results emphasize the difference between CD38 and other enzymes in which the cADPR cyclase activity predominates.     

Determination of inhibition constants, I50 values and the type of inhibition for enzyme-catalyzed reactions

A procedure is proposed for determining whether an inhibitor of an enzyme-catalyzed reaction is competitive, noncompetitive, or uncompetitive with respect to the substrate. The method is based on fitting the equation for noncompetitive inhibition to data obtained by measuring the rate of the reaction over a range of substrate and inhibitor concentrations. The results of this fit may suggest that the inhibition may be either competitive or uncompetitive, whereupon the data are reanalyzed using the appropriate equation. Comparison of this second fit with the first using an F test permits a statistical decision to be made on the type of inhibition. The chosen fit yields values and standard errors for the Michaelis-Menten parameters (maximum velocity and Michaelis constant), as well as the inhibition constant(s). From these values it is then possible to predict the I50, and its standard error, at any chosen substrate concentration, thereby facilitating comparison with results obtained with similar inhibitors, for homologous enzymes, or in other laboratories.     

Oxygen dependent lactate utilization by Lactobacillus plantarum

Lactobacillus plantarum P5 grew aerobically in rich media at the expense of lactate; no growth was observed in the absence of aeration. The oxygen-dependent growth was accompanied by the conversion of lactate to acetate which accumulated in the growth medium. Utilization of oxygen with lactate as substrate was observed in buffered suspensions of washed whole cells and in cell-free extracts. A pathway which accounts for the generation of adenosine triphosphate during aerobic metabolism of lactate to acetate via pyruvate and acetyl phosphate is proposed. Each of the enzyme activities involved, nicotinamide adenine dinucleotide independent lactic dehydrogenase, nicotinamide adenine dinucleotide dependent lactic dehydrogenase, pyruvate oxidase, acetate kinase and NADH oxidase were demonstrated in cell-free extracts. The production of pyruvate, acetyl phosphate and acetate was demonstrated using cell-free extracts and cofactors for the enzymes of the proposed pathway.Abbreviations MRS Man, Rogosa and Sharpe (1960) medium modified as in Materials and methods - TY Tryptone Yeast Extract broth - OUL Oxygen uptake with lactate as substrate - DCPIP 2,6-Dichlorophenolindophenol - LDH Lactic dehydrogenase     

Intra- and intermolecular electron transfer processes in redox proteins

Initial velocity and product inhibition experiments were performed to characterize the kinetic mechanism of branched chain ketoacid dehydrogenase (the branched chain complex) activity. The results were directly compared to predicted patterns for a three-site ping-pong mechanism. Product inhibition experiments confirmed that NADH is competitive versus NAD+ and isovaleryl CoA is competitive versus CoA. Furthermore, both NADH and isovaleryl CoA were uncompetitive versus ketoisovaleric acid. These results are consistent with a ping-pong mechanism and are similar to pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. However, inhibition patterns for isovaleryl CoA versus NAD+ and NADH versus CoA are not consistent with a ping-pong mechanism. These patterns may result from a steric interaction between the flavoprotein and transacetylase subunits of the complex. To determine the kinetic mechanism of the substrates and feedback inhibitors (NADH and isovaleryl CoA) of the branched chain complex, it was necessary to define the interaction of the inhibitors at nonsaturating fixed substrate (CoA and NAD+) concentrations. While the competitive inhibition patterns were maintained, slope replots for NADH versus NAD+ at nonsaturating CoA concentrations were parabolic. This unexpected finding resembles a linear mixed type of inhibition where the inhibition is a combination of pure competitive and noncompetitive inhibition.     

Combined coenzyme-substrate analogues of various dehydrogenases. Synthesis of (3S)- and (3R)-5-(3-carboxy-3-hydroxypropyl)nicotinamide adenine dinucleotide and their interaction with (S)- and (R)-lactate-specific dehydrogenases.

Two diastereomeric nicotinamide adenine dinucleotide (NAD+) derivatives were synthesized in which the substrates of (S)-and (R)-lactate-specific dehydrogenases are covalently attached via a methylene spacer at position 5 of the nicotinamide ring. The corresponding nicotinamide derivatives were obtained stereospecifically by enzymatic reduction of 5-(2-oxalylethyl)nicotinamide. (3S)-5-(3-Carboxy-3-hydroxypropyl)-NAD+ undergoes and intramolecular hydride transfer in the presence of pig heart lactate dehydrogenase, forming the corresponding coenzyme-substrate analogue composed of pyruvate and NADH. No cross-reaction products resulting from an intermolecular reaction are observed. Two (R)-lactate specific dehydrogenases, however, do not catalyze a similar reaction in either one of the two diastereomers. A possible arrangement of the substrates in the active centers of these enzymes is proposed. 5-Methyl-NAD+ and 5-methyl-NADH are active coenzymes of pig heart lactate dehydrogenase in contrast to reports in the literature. (S)-Lactate binds to this enzyme in the absence of coenzyme, exhibiting a dissociation constant of 11 mM.     

Purification and properties of pyridine nucleotide-independent L-lactate dehydrogenase from Polyporus circinatus

Cell extracts of Polyporus circinatus grown on lactate catalyze the reduction of 2,6-dichlorophenolindophenol by l-lactate without the participation of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate. The enzyme has been purified 78-fold and was homogenous by disc gel electrophoresis. The optimal pH was found to be 6.7. The Michaelis constant for l-lactate was 5.9 x 10(-4) M and the oxalate inhibition constant was 1.5 x 10(-4) M. The nature of the prosthetic group is discussed.     

Coupled-Enzyme System for Measuring Viral Neuraminidase Activity

A coupled-enzyme assay for determining viral neuraminidase activity is described. All reactants-viral neuraminidase, the initial substrate (fetuin), N-acetylneuraminic acid aldolase, lactic acid dehydrogenase, and reduced nicotinamide adenine dinucleotide-are combined in a single cuvette. Thus, in a single coupled system neuraminidase releases N-acetylneuraminic acid, which is cleaved to N-acetyl-D-mannosamine and pyruvic acid; finally, pyruvate is reduced to lactate as reduced nicotinamide adenine dinucleotide is oxidized. The rate of change of absorbance at 340 nm, as reduced nicotinamide adenine dinucleotide is oxidized, is a measure of the rate of reaction of the coupled system. This procedure, which measures the rate of release of N-acetylneuraminic acid by neuraminidase, is an alternate method for those procedures which require multistep, colorimetric determinations.     

Calculation of inhibitor Ki and inhibitor type from the concentration of inhibitor for 50% inhibition for Michaelis-Menten enzymes

The use of I50 (concentration of inhibitor required for 50% inhibition) for enzyme or drug studies has the disadvantage of not allowing easy comparison among data from different laboratories or under different substrate conditions. Modifications of the Michaelis-Menten equation for treatment of inhibitors can allow both the determination of the type of inhibition (competitive, noncompetitive, and uncompetitive) and the Ki for the inhibitor. For competitive and uncompetitive inhibitors when the assay conditions are [S] = Km, then Ki = I50/2. For different conditions of [S] there is a divergence between competitive and uncompetitive inhibitors that may be used to identify the type of inhibitor. The equation for Ki also differs. For noncompetitive inhibitors the Ki = I50 and this relationship is valid with changing [S]. The equations developed require a single substrate, reversible-type inhibitors, and kinetics of the Michaelis-Menten type. Examples of the use of the equations are illustrated with experimental data from scientific publications.     

A kinetic study of the alpha-keto acid dehydrogenase complexes from pig heart mitochondria.

The kinetic mechanisms of the 2-oxoglutarate and pyruvate dehydrogenease complexes from pig heart mitochondria were studied at pH 7.5 and 25 degrees. A three-site ping-pong mechanism for the actin of both complexes was proposed on the basis of the parallel lines obtained when 1/v was plotted against 2-oxoglutarate or pyruvate concentration for various levels of CoA and a level of NAD+ near its Michaelis constant value. Rate equations were derived from the proposed mechanism. Michaelis constants for the reactants of the 2-oxoglutarate dehydrogenase complex reaction are: 2-oxoglutarate, 0.220 mM; CoA, 0.025 mM; NAD+, 0.050 mM. Those of the pyruvate dehydrogenase complex are: pyruvate, 0.015 mM; CoA, 0.021 mM; NAD+, 0.079 mM. Product inhibition studies showed that succinyl-CoA or acetyl-CoA was competitive with respect to CoA, and NADH was competitive with respect to NAD+ in both overall reactions, and that succinyl-CoA or acetyl-CoA and NADH were uncompetitive with respect to 2-oxoglutarate or pyruvate, respectively. However, noncompetitive (rather than uncompetitive) inhibition patterns were observed for succinyl-CoA or acetyl-CoA versus NAD+ and for NADH versus CoA. These results are consistent with the proposed mechanisms.     

Alternative-substrate inhibition of L-lactate transport via the monocarboxylate-specific carrier system in human erythrocytes

The L-lactate/proton symport system of the red blood cell membrane was studied under conditions of alternative-substrate inhibition by glycolate. At constant pH of the medium glycolate caused competitive inhibition of L-lactate transport. In Lineweaver-Burk plots of 1/v against 1/[H], on the other hand, glycolate caused an uncompetitive inhibition. These observations indicate, that the monocarboxylate carrier exhibits ordered substrate binding, with the proton binding first.     

Interactions of nicotinamide-adenine dinucleotide phosphate analogues and fragments with pigeon liver malic enzyme. Synergistic effect between the nicotinamide and adenine moieties.

The structural requirements of the NADP+ molecule as a coenzyme in the oxidative decarboxylation reaction catalysed by pigeon liver malic enzyme were studied by kinetic and fluorimetric analyses with various NADP+ analogues and fragments. The substrate L-malate had little effect on the nucleotide binding. Etheno-NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, and nicotinamide-hypoxanthine dinucleotide phosphate act as alternative coenzymes for the enzyme. Their kinetic parameters were similar to that of NADP+. Thionicotinamide-adenine dinucleotide phosphate, 3-aminopyridine-adenine dinucleotide phosphate, 5'-adenylyl imidodiphosphate, nicotinamide-adenine dinucleotide 3'-phosphate and NAD+ act as inhibitors for the enzyme. The first two were competitive with respect to NADP+ and non-competitive with respect to L-malate; the other inhibitors were non-competitive with NADP+. All NADP+ fragments were inhibitory to the enzyme, with a wide range of affinity, depending on the presence or absence of a 2'-phosphate group. Compounds with this group bind to the enzyme 2-3 orders of magnitude more tightly than those without this group. Only compounds with this group were competitive inhibitors with respect to NADP+. We conclude that the 2'-phosphate group is crucial for the nucleotide binding of this enzyme, whereas the carboxyamide carbonyl group of the nicotinamide moiety is important for the coenzyme activity. There is a strong synergistic effect between the binding of the nicotinamide and adenosine moieties of the nucleotide molecule.     

Structure determination and refinement of Bacillus stearothermophilus lactate dehydrogenase

Structures have been determined of Bacillus stearothermophilus "apo" and holo lactate dehydrogenase. The holo-enzyme had been co-crystallized with the activator fructose 1,6-bisphosphate. The "apo" lactate dehydrogenase structure was solved by use of the known apo-M4 dogfish lactate dehydrogenase molecule as a starting model. Phases were refined and extended from 4 A to 3 A resolution by means of the noncrystallographic molecular 222 symmetry. The R-factor was reduced to 28.7%, using 2.8 A resolution data, in a restrained least-squares refinement in which the molecular symmetry was imposed as a constraint. A low occupancy of coenzyme was found in each of the four subunits of the "apo"-enzyme. Further refinement proceeded with the isomorphous holo-enzyme from Bacillus stearothermophilus. After removing the noncrystallographic constraints, the R-factor dropped from 30.3% to a final value of 26.0% with a 0.019 A and 1.7 degrees r.m.s. deviation from idealized bond lengths and angles, respectively. Two sulfate ions per subunit were included in the final model of the "apo"-form--one at the substrate binding site and one close to the molecular P-axis near the location of the fructose 1,6-bisphosphate activator. The final model of the holo-enzyme incorporated two sulfate ions per subunit, one at the substrate binding site and another close to the R-axis. One nicotinamide adenine dinucleotide coenzyme molecule per subunit and two fructose 1,6-bisphosphate molecules per tetramer were also included. The phosphate positions of fructose 1,6-bisphosphate are close to the sulfate ion near the P-axis in the "apo" model. This structure represents the first reported refined model of an allosteric activated lactate dehydrogenase. The structure of the activated holo-enzyme showed far greater similarity to the ternary complex of dogfish M4 lactate dehydrogenase with nicotinamide adenine dinucleotide and oxamate than to apo-M4 dogfish lactate dehydrogenase. The conformations of nicotinamide adenine dinucleotide and fructose 1,6-bisphosphate were also analyzed.