Abortive Ternary Complex Research Articles

Studies on enzymes from parasitic helminths—III. Purification and properties of lactate dehydrogenase from the tapeworm, Hymenolepis diminuta

1. A procedure for the purification of lactate dehydrogenase (E.C. 1.1.1.27) from the tapeworm, Hymenolepis diminuta, is described. 2. The 128-fold purified enzyme exhibits a specific activity of 106 units/mg of protein and was adjudged to be homogeneous by rechromatography, sedimentation velocity and sedimentation equilibrium ultracentrifugation, and a variety of electrophoretic techniques. 3. Only one form of the enzyme is present in the tapeworm as demonstrated by electrophoresis on cellulose acetate, polyacrylamide-gel and isoelectric focusing. 4. The molecular weight of the native enzyme is 141,000 and exhibits a sedimentation coefficient of 6·89 × 10 -13 sec. The enzyme is a tetramer composed of subunits of molecular weight 36,000. However, under low protein concentrations (0·1 mg/ml) on analytical gel filtration, the enzyme dissociates to the dimeric state and exhibits a molecular weight of 75,000. 5. Inhibitor studies are consistent with an active center which may be catalytically and structurally similar to the enzyme from vertebrates. 6. The enzyme is kinetically similar to vertebrate heart LDH. The K m for pyruvate was 0·17 mM and for lactate was 6·3 mM. The H. diminuta lactate dehydrogenase also forms an abortive ternary complex with coenzyme and substrate. 7. The data are discussed in regard to the physiology of the parasite and the host.

Coenzyme binding to glutamate dehydrogenase. A study by relaxation kinetics.

1 The binding of NADH and NADPH to ox liver glutamate dehydrogenase have been studied by the temperature-jump technique using fluorescence detection. 2 The rate of combination of NADH with glutamate dehydrogenase is considerably slower than if it were diffusion controlled while the rate constant for dissociation is too fast for this to be a rate-limiting step in the overall catalytic reaction. 3 The binding constant obtained rom the ratio of the rate constants agrees well with a value previously determined by equilibrium dialysis. 4 Comparison of NADH with NADPH strongly suggests that binding to a site common to both coenzymes is being studied and this must be the active site. 5 The inhibitor guanosine 5′-triphosphate and the activator adenosine 5′-diphosphate both affect the association and dissociation rate constants but not sufficiently to account for the observed steady-state inhibition and activation. 6 In the presence of the substrate l-glutamate a second slower relaxation time is seen and is ascribed to the formation of an abortive ternary complex. This step is not observed with the poorer substrate l-alanine. 7 The data enable a model of the relationship between the active site and the polymerising site to be proposed.

Active centre equivalent weight of glutamate dehydrogenase from dry weight determinations and spectrophotometric titrations of abortive complexes

The extinction coefficient of bovine liver glutamate dehydrogenase ( l-glutamate:NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3) at 280 nm has been redetermined from dry weight estimations as 0.93 cm 2· mg −1 . Glutamate and glutarate form abortive ternary complexes with the enzyme and NAD(P)H, the absorption spectra of which differ considerably from those of the free coenzymes and their binary enzyme complexes, and permit estimation of the equivalent weight of the active centre by spectrophotometric titration. A value of 57 000 is obtained.

The formation of ternary complexes by diphosphopyridine nucleotide-dependent dehydrogenases

Formation of abortive ternary complexes between enzyme, coenzyme, and substrate has been demonstrated for several pyridine nucleotide-dependent dehydrogenases by measuring the quenching of protein fluorescence. Evidence is presented suggesting the participation of a ternary complex consisting of lactate dehydrogenase, DPN +, and pyruvate in the phenomenon of substrate inhibition observed with lactate dehydrogenases. This complex is more readily formed with the H-type LDH than with the M-type enzyme under identical conditions. The ternary complex of LDH-DPN +-pyruvate can be formed at physiological levels of pyruvate. Information concerning the structure of the ternary complex has been derived from its absorption and fluorescence spectra, as well as from its kinetic and chemical properties. The pyridine ring in the complex appears to be in the reduced form, similar to that found in DPNH. The ternary complex can be precipitated with ammonium sulfate. Denaturation of the enzyme results in a product which is easily oxidized. Evidence is presented indicating that this product consists of DPN which is covalently bonded to pyruvate. Similar products can be obtained with analogs of DPN, but the reaction for lactate dehydrogenase is specific for pyruvate. Related ternary complexes have been observed from malate dehydrogenase, glutamic dehydrogenase, and alcohol dehydrogenase. The significance of ternary complex formation is discussed in relation to the reaction mechanism of pyridine nucleotide-linked dehydrogenases.

The 5Åresolution structure of an abortive ternary complex of lactate dehydrogenase and its comparison with the apo-enzyme

A low resolution (5.0Å) three-dimensional electron density map of the abortive ternary complex of dogfish muscle (M 4) lactate dehydrogenase, NAD and pyruvate has been calculated. The tetramer of the abortive ternary complex, like that of the apo-enzyme, has 222 symmetry. The two crystal structures, however, show totally different packing of tetramers. The major sites of heavy atom substitution correspond with some of the apo-enzyme sites. The main-chain conformation of lactate dehydrogenase in the abortive ternary complex is very similar to that of the apo-enzyme except for one part of the chain close to the coenzyme binding site. This piece of chain, which stands out in the form of a closed loop in the apo-enzyme, folds down over the active site cavity in the ternary complex. The residues at the very top of this loop move by as much as 12Åtowards the active center cavity. The coenzyme molecule in the ternary complex is observed in the same position as it was found by diffusion of NAD into apo-enzyme crystals.

The kinetics of yeast hexokinase in the light of the induced fit involved in the binding of its sugar substrate.

The kinetics of the initial reaction rates of yeast hexokinase, both in the forward and in the backward direction, have been re‐examined in the light of recent findings concerning the properties of the ATPase activity of hexokinase and its modification by certain non‐phosphorylable analogues of the sugar substrates.A single pattern has been found in the double reciprocal plots even if the concentrations of both the acceptor and the donor substrates are varied over a wide range, with ITP as well as with ATP as donor substrates. A similar pattern has been found for the backward reaction.These results are interpreted as indicative that in the forward as well as in the backward reaction, the interconversion of the ternary complex of the enzyme with both substrates is the limiting step. This fact and the complex effect of glucose 6‐phosphate as an initially‐added product, prevents in principle the distinction between the possible kinetic mechanisms leading to the ternary complex. Nevertheless, taking into account the change in affinities for the nucleotide substrates specifically induced by certain sugars, it becomes possible to formulate a plausible hypothesis. It is postulated that binding of a sugar to an enzyme‐nucleotide complex results in an abortive ternary complex. Accordingly, formation of active ternary complexes in the hexokinase reaction requires the sequential addition of a nucleotide substrate to a binary complex of the enzyme with a sugar substrate.

Alkylation studies on a reactive histidine in pig heart malate dehydrogenase.

1 From the pH dependence of the rate of alkylation of pig heart malate dehydrogenase by iodoacetamide the pKa of the reactive histidine residue was determined as 7.1. 2 The enzyme is not inactivated by iodoacetic acid or 3-bromopropionic acid. 3 NADH protects the enzyme almost completely against alkylation by iodoacetamide whereas NAD, ADP-ribose and N-methylnicotinamide give only partial protection. 4 Oxaloacetate and malate do not protect against alkylation but mixtures of oxaloacetate and NAD or ADP-ribose protect completely, demonstrating formation of abortive ternary complexes.

Studies on Rates of Abortive Ternary Complex Formation of Lactate Dehydrogenase Isozymes

Abstract Rates of formation and dissociation of an abortive ternary complex between lactate dehydrogenase isozymes from various mammalian tissues, NAD+, and pyruvate were studied utilizing stopped-flow spectrofluorometry. Formation of the complex was accompanied by a decrease in protein fluorescence at 340 mµ and a loss of lactate dehydrogenase activity. Rates of formation of an abortive lactate dehydrogenase-1 and lactate dehydrogenase-5 complex were compared at 25° and 40° over a range of pyruvate and enzyme concentrations, at two NAD+ concentrations, and with two coenzyme analogues. Dialysis against buffer produced an increase in protein fluorescence and recovery of lactate dehydrogenase activity that suggested dissociation of the abortive complex. At pyruvate concentrations of 0.3 mm the abortive ternary complex formed twice as fast with lactate dehydrogenase-1 as with lactate dehydrogenase-5 and with 40.0 mm pyruvate it formed 5 times faster. The rate of complex formation increased with both isozymes when pyruvate concentrations were raised from 0.3 mm to 40.0 mm and when the temperature was elevated from 25° to 40°. However, the rates of abortive complex formation were inversely related to enzyme concentration. Compared to lactate dehydrogenase turnover numbers, the rates of formation of the abortive ternary complex were slow. Under physiological conditions of temperature (40°) and pyruvate concentration (0.3 to 1.0 mm), half the maximum fluorescence quenching required approximately 50 sec for partially purified lactate dehydrogenase-1. Changes in coenzyme concentration from 0.10 mm to 0.75 mm failed to alter the rates of abortive complex formation. With the 3-acetylpyridine analogue of NAD+, rates of abortive complex formation for both lactate dehydrogenase-1 and lactate dehydrogenase-5 were similar, whereas with deamino-NAD+ the abortive complex formed faster with lactate dehydrogenase-1 than with lactate dehydrogenase-5. Dialysis of the abortive complex resulted in recovery of enzyme activity; the rate of recovery was faster with lactate dehydrogenase-5 than with lactate dehydrogenase-1. The stopped-flow spectrophotofluorometer permitted measurement of lactate dehydrogenase activity at enzyme concentrations approaching those in tissue. At lactate dehydrogenase-1 concentrations of 3.5 x 10-6 m or 3.5 x 10-7 m no substrate inhibition was detected with pyruvate concentrations up to 20.0 mm, whereas with enzyme levels at and below 1.8 x 10-7 m, substrate inhibition appeared at pyruvate concentrations greater than 1.0 mm.

Crystalline ternary complexes of lactate dehydrogenase

An alternative space group of an abortive ternary complex of dogfish M4 † lactate dehydrogenase:NAD-pyruvate has been found. 5 Å resolution X-ray diffraction data have been analyzed and the resulting difference electron density maps indicate the loss of coenzyme substitution in two of the four subunits.

Inosinic Acid Dehydrogenase of Sarcoma 180 Cells

Abstract Inosinic acid dehydrogenase was partially purified from sarcoma 180 cells. The concentrations of each reactant, inosine 5'-phosphate, nicotinamide adenine dinucleotide, and K+, altered both the apparent Km and Vmax values for each of the other reactants. Enzymatic activity was inhibited by both products of the reaction, xanthosine 5'-phosphate and reduced nicotinamide adenine dinucleotide; the inhibition produced by XMP was competitive with respect to IMP, whereas the inhibition caused by NADH was not competitive with respect to either IMP, NAD+, or K+. The data are consistent with an enzymatic mechanism involving ordered sequential addition of IMP, NAD+, and K+ to the enzyme to form active enzyme-substrate complexes. Substrate inhibition occurred at NAD+ concentrations several-fold higher than the optimal concentration of NAD+; the kinetics suggests that formation of an abortive ternary complex between the enzyme, XMP, and NAD+ is involved in producing this substrate inhibition.

Spectrophotometric analysis of rat liver lactate dehydrogenase-coenzyme and coenzyme analog complexes

A study of difference spectra in the range, 300–500 mμ is reported involving highly purified rat liver lactate dehydrogenase (LDH) and oxidized or reduced coenzyme or coenzyme analog (nicotinamide-adenine dinucleotide (NAD) or acetylpyridine-adenine dinucleotide (AcPyAD). The formation of the binary complexes, LDH · NAD or LDH · AcPyAD is associated with the development of spectral bands with λ max at 335 mμ and 355 mμ, respectively. The observed absorbance is proportional to coenzyme concentration at fixed LDH concentration. The spectral behavior of the LDH · oxidized coenzyme binary complexes is clearly distinguishable from that of the binary complexes involving NADH or AcPyADH. A conclusion is that the binding of oxidized coenzymes to LDH results in a significant change in the chromophoric behavior of a hybridized form of the substituted pyridine ring. The formation of abortive ternary complexes involving LDH · oxidized coenzyme /sd pyruvate or LDH · oxidized coenzyme · /ga-ketobutyrate was accompanied by slow progressive spectral changes which reached equilibrium values, dependent on the concentration of all three reactants. For LDH · NAD · pyruvate λ max, was 323 mμ; for LDH · AcPyAD · pyruvate λ max was 341 mμ. The spectral changes were fully reversible upon dialysis vs. AcPyAD-buffer in the one case but not upon dialysis vs. NAD-buffer in the other. Complete recovery of enzyme activity was noted in each case after dialysis to remove pyruvate or α-ketobutyrate. The spectral changes are considered to be those associated with the allosteric binding of pyruvate or α-ketobutyrate to binary complexes of LDH · oxidized coenzyme with resulting conformational changes in LDH which affect the active sites. No abortive ternary complex involving LDH-reduced coenzyme (or reduced coenzyme analog) -lactate could be detected by spectral means.

Malate Dehydrogenases: III. ALTERATION OF CATALYTIC PROPERTIES DURING PURIFICATION OF BACILLUS SUBTILIS MALATE DEHYDROGENASES

Abstract The apparent pH optimum of oxalacetate reduction by the malate dehydrogenase of Bacillus subtilis changed from pH 9.0 to pH 6.0 during the three crystallizations required to achieve maximal specific activity. The change was apparently due to an alteration in the substrate inhibition characteristics of the enzyme. The degree of substrate inhibition at a given oxalacetate concentration was dependent upon the pH of the assay mixture, and was greater for the than for the crystallized preparations at each pH value studied. In the presence of excess oxalacetate at pH 9.0, the impure enzyme exhibited slight substrate inhibition, while the crystallized enzyme exhibited augmented activity with an increase in oxalacetate concentration. Some differences were also observed between the two forms with respect to their reactions with coenzyme analogues and malate. Conditions leading to unfolding or dissociation or both of the protein brought about transient changes in the substrate inhibition properties of the impure enzyme, but did not alter these characteristics of the crystallized enzyme. No conditions were found which would permanently convert either form of the enzyme into the other. The spectrum of an abortive ternary complex formed by the crystallized enzyme with oxidized substrate (oxalacetate) and oxidized cofactor (acetylpyridine analogue of diphosphopyridine nucleotide) is also presented.

Determination of Dissociation Constants of Coenzymes and Abortive Ternary Complexes with Rabbit Muscle Lactate Dehydrogenase from Fluorescence Measurements

Determination of Dissociation Constants of Coenzymes and Abortive Ternary Complexes with Rabbit Muscle Lactate Dehydrogenase from Fluorescence Measurements