Dogfish M4 Lactate Dehydrogenase Research Articles

Structure determination and refinment of Bacillus stearothermophilus lactate dehydrogenase

AbstractStructures have been determined of Bacillus stearothermophilus “apo” and holo lactate dehydrogenase. The holo‐enzyme had been co‐crystallized with the activator fructose 1,6‐biosphosphate. 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 Å resolution by means of the noncrystallographic molecular 222 symmetry. The R‐factor was reduced to 28.7%, using 2.8 Å resolution data, in a restrained least‐squares refnement in which the molecula rsymmetry 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‐enzyume 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 Å and 1.7° r.m.s. deviation from idealized bond length and angles, respectively.Two sulfate ions per subunit were included in the final model of the “apo” ‐from‐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 incotinamide 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.

Simulation of the enzymatic reaction of dogfish M4 lactate dehydrogenase: a molecular orbital study on the reactivity of pyruvate.

Simulation of the enzymatic reaction of dogfish M4 lactate dehydrogenase (LDH) was performed. In the enzymatic reaction from L-lactate to pyruvate, the structure in which the proton transfer from L-lactate to His 195 is coupled with the hydride transfer from L-lactate to NAD+ was simulated. In the reverse reaction, the structure in which the proton transfer from His 195 to pyruvate is coupled with the hydride transfer from NADH to pyruvate was also simulated. Since the proton of His 195 approaches the carbonyl oxygen of pyruvate from the π direction of the pyruvate plane in the simulation, the electronic structure and the reactivity of pyruvate were studied from a quantum chemical point of view, using the ab initio LCAO MO method. When the carbonyl carbon of pyruvate is attacked by the hydride ion of NADH, the total π electron density of the carbonyl oxygen of pyruvate increases substantially. Therefore, the proton of His 195 attacks the carbonyl oxygen of pyruvate from the π direction. Moreover, the pyruvate-Arg 171 complex having two nearly parallel hydrogen bonds is more easily attacked by hydride than complexes having other conformations.

Structural adaptations of lactate dehydrogenase isozymes.

The three-dimensional structures of dogfish M4 (muscle) and pig H4 (heart) lactate dehydrogenase (L-lactate:NAD+ oxidoreductase, EC 1.1.1.27) have been determined and correlated with the amino acid sequences of the dogfish M4, pig M4, pig H4, chicken M4, and chicken H4 lactate dehydrogenase isozymes. These results have been related to the known differences of physicochemical properties between the M and H lactate dehydrogenase isozymes. By far the largest structural alterations occur in the transition between the "apo" and "ternary complex" conformational states of the enzyme rather than between species or isozymes. The major catalytic difference can be explained by a replacement of alanine (in the M chain) with a glutamine (in the H chain) in the vicinity of the binding site of the coenzyme phosphates. The known immunological differentiation of the M and H isozymes is consistent with the differences in their amino acid sequences.

Dogfish M4 lactate dehydrogenase: reversible inactivation by pyridoxal 5′-phosphate and complete protection in complexes that mimic the active ternary complex

Dogfish M4 lactate dehydrogenase, like the corresponding pig enzyme, is inactivated by pyridoxal 5'-phosphate through modification of a single essential lysine residue. The activity is completely protected in the complexes E-NAD+-oxalate, E-NADH-oxamate and E-(NAD+-pyruvate adduct), but only partially protected in E-NAD+, E-NADH, E-NAD+-oxamate and E-NADH-oxalate.

The amino acid sequence of the tryptic peptides isolated from dogfish M4 lactate dehydrogenase.

The peptides which result from treatment of the S-[14C]carboxymethyl derivative of dogfish M4 lactate dehydrogenase (EC 1.1.1.27) with trypsin have been isolated and their sequences have been elucidated. Each identical subunit has a molecular weight of 36,000 and on the basis of the amino acid composition 40 unique tryptic peptides are anticipated. Thirty-seven of these peptides have been isolated and have been completely characterized. The amino acid sequences of these peptides are presented.

Aminoacid Sequence of Dogfish M 4 Lactate Dehydrogenase

Aminoacid Sequence of Dogfish M ₄ Lactate Dehydrogenase

Aminoacid sequence of dogfish M4 lactate dehydrogenase.

About 80% of the aminoacid sequence of dogfish (Squalus acanthius) M(4) lactate dehydrogenase (EC 1.1.1.27) has been elucidated. Several sequence homologies with peptides from pig H(4) and pig M(4) lactate dehydrogenase are identified. Histidine 195 is homologous to the essential histidine residue in pig H(4) lactate dehydrogenase. Similarities in the sequence around the "essential" cysteine residue of lactate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and yeast and liver alcohol dehydrogenase are delineated.

Physical and Chemical Properties of Reversibly Inactivated Lactate Dehydrogenases

Abstract The effect of reversible inactivation on the physical and chemical properties of dogfish M4 and chicken heart H4 lactate dehydrogenases has been studied. Reduced diphosphopyridine nucleotide was found to have a significant effect on the rate of recovery of the enzymatic activity, but not on the over-all recovery. Optical rotatory dispersion, circular dichroism, fluorescence, and immunological studies have shown structural differences between the native and reassociated enzyme. Further evidence for the difference in conformational structure was obtained from the pattern of reactivity of sulfhydryl groups of the reactivated enzyme. The native enzyme was also found to be more stable to heat and urea denaturation than the reassociated form. Pyruvate inhibition, and the catalytic activity with coenzyme analogues, was found to be different for the native and the reassociated enzyme. These results indicate the possibility that a conformational difference exists between the native and the reassociated enzyme for both dogfish M4 and chicken H4 lactate dehydrogenase. Studies on the lactate dehydrogenase reassociated in the presence of reduced DPN showed structural characteristics which are closer to the native dehydrogenase than the enzyme which was reassociated in the absence of the reduced coenzyme. Incubation of the enzyme with DPNH after reassociation does not convert the reassociated enzyme to a structure similar to that of the native enzyme. These studies suggest a possible role of the reduced coenzyme in the folding of the peptide chain or in the subunit interaction and in the attainment of the native conformation of the enzyme.