Margaret J. Adams

A comparison of the structures of apo dogfish M4 lactate dehydrogenase and its ternary complexes.

Abstract Details are recorded of the X-ray diffraction data collection, heavy atom refinement and preliminary structure refinement for two different dogfish M4 lactate dehydrogenase structures. One of these is the 2.0 A resolution apoenzyme structure; the other is a 3.0 A resolution abortive ternary complex. Two other ternary substrate inhibitory complexes (LDHase † : NAD: oxalate and LDHase: NADH: oxamate), isomorphous with the abortive ternary complex (LDHase: NAD-pyruvate), have also been examined. The apo-LDHase and LDHase: NAD-pyruvate structures are systematically compared to determine significant differences in their conformation. These are related to differences in structure amongst the three studied ternary complexes. These differences all occur in regions of the protein around the active site, particularly the flexible loop covering the active center pocket and the C-terminal helix αH. The changes are suggestive of a domino effect whereby the closing of the loop on binding coenzyme and substrate triggers the critical reactive residues into assuming their catalytically active positions.

Low resolution study of crystalline l-lactate dehydrogenase

The electron density distribution of dogfish muscle lactate dehydrogenase, based on 2000 independent terms extending to 5 A resolution, shows the shape of the tetrameric molecule and of the individual subunits. Each of the five heavy-atom derivatives used in the calculation substitutes at one or more of three sites A, B and C. The heavy-atom compounds (sodium p-hydroxymercuribenzoate, dimercury acetate, Baker dimercurial, platinum ethylene diamine dichloride, and sodium aurichloride) all react with a sulphydryl group at A to some extent. Sodium aurichloride substitutes at site B with some loss of isomorphism, while the platinum compound occupies site C. Anomalous dispersion measurements have been used to determine the absolute configuration of the molecule and to improve phasing. A second electron density map includes 2000 extra terms which have been phased using only two of these compounds, extending the resolution to approximately 4 A. It is possible to trace only some parts of the polypeptide chain of the subunits in either map since the molecule does not appear to have any distinctive secondary structure. Adenosine, a competitive inhibitor, has been found to bind close to the “nonessential” thiol at site A, at the surface between adjoining subunits. The subunit is divided into two parts by a narrower “neck”; the “essential” thiol group is at site B in this region. On diffusion of the coenzyme, nicotinamide adenine dinucleotide, into grown crystals, the crystal symmetry is lowered; the modification is probably caused by a change in quaternary structure. A similar change is caused by certain parts of the coenzyme.

Conformation of coenzyme fragments when bound to lactate dehydrogenase

Abstract The conformations of adenosine, 5′-AMP and 5′-ADP when bound to dogfish M 4 lactate dehydrogenase at pH 7.8 or greater have been determined at 2.8 A resolution to investigate the events on coenzyme binding. The coenzyme fragments AMP and ADP induce a conformational change in lactate dehydrogenase at pH values less than 6.0 in the same way as do NAD + , NADH or ADPR at any pH value. The structure of NAD + when bound to lactate dehydrogenase had previously been determined at 5.0 A resolution. The structures of the bound adenosine, AMP, ADP and NAD + are compared with the preliminary structure of NAD in a 3.0 A resolution map of the ternary complex LDH-NAD—pyruvate. Small but significant changes in the binding of the phosphates could be important in the folding of the protein loop over the substrate binding pocket.

Molecular symmetry axes and subunit interfaces in certain dehydrogenases

Abstract A right-handed orthogonal set of axes P , Q , R has been denned coincident with the three different molecular 2-fold axes of lactate dehydrogenase. The four different symmetry-related subunits can then be color-coded red, yellow, green and blue to identify the three different subunit contacts made by any one subunit to the remaining three others. This nomenclature has been extended to soluble malate dehydrogenase and will facilitate the comparison of dehydrogenases with related ternary and quaternary structures.

The structure of the nicotinamide-adenine dinucleotide coenzyme when bound to lactate dehydrogenase.

The electron density distribution of M4 dogfish lactate dehydrogenase at 5 A resolution has been re-calculated using improved phases derived in part from a new heavy atom derivative. Difference maps have been calculated for an adenosine and a nearly isomorphous NAD derivative, permitting the positioning of the coenzyme on the molecule and identification of its adenosine moiety. A skeletal model has been fitted to the electron density which clearly establishes the “open” structure of the bound coenzyme. The coenzyme is bound at the base of a deep cleft with the nicotinamide end buried particularly deeply within the subunit. The reactive (A-type) hydrogen comes within 13.5 A of the essential thiol group, but is separated from it by substantial protein electron density. No two of the four coenzyme binding sites approach closer than 21 A.

Functional anion binding sites in dogfish M4 lactate dehydrogenase

X-ray diffraction data have been collected from dogfish M4 lactate dehydrogenase crystals in which ammonium sulfate had been exchanged by citrate at pH 6.0 and 7.8. Data were also collected from crystals which had been soaked in 0.1 m oxamate, a lactate dehydrogenase inhibitor. The difference electron density maps obtained have been interpreted in terms of two exchangeable anion binding sites, one at the active center and one between two subunits. The active center site is coincident with the substrate binding site in a ternary complex, while the subunit boundary site, which has been observed in several different forms of the enzyme, may be involved in stabilizing the tetramer.

Atomic co-ordinates for dogfish M4 apo-lactate dehydrogenase.

Abstract Atomic co-ordinates for one subunit of LDH are given in A with respect to a Cartesian co-ordinate system corresponding to the molecular two-fold axes.

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

Abstract A low resolution (5.0A) three-dimensional electron density map of the abortive ternary complex of dogfish muscle (M4) 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 12Atowards 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.

Structure of 6-phosphogluconate dehydrogenase from sheep liver at 6 Å resolution

A 6 A resolution electron density map has been calculated for 6-phosphogluconate dehydrogenase from sheep liver. The dimeric enzyme crystallizes with a single subunit in the asymmetric unit. The structure was determined by the method of isomorphous replacement. The two heavy-atom derivatives used were obtained by soaking crystals in potassium dicyanoaurate, which bound at two sites, and potassium tetracyanoplatinate, which bound at three different sites in the monomer. The resulting electron density map shows the molecular boundary and indicates which of the crystallographic 2-fold axes is the molecular 2-fold. The subunit is ellipsoidal with a large indentation distant from the subunit boundary. Several columns of density of the correct dimensions for α-helices can be seen. Some of these are a part of the subunit contact area and two are more than 40 A units long.

A crystalline form of testes-specific lactate dehydrogenase

Abstract A preliminary X-ray diffraction examination of crystals of lactate dehydrogenase from mouse testes (LDH-X) showed these to be triclinic. The one molecule per unit cell occupies a similar crystal volume to other forms of lactate dehydrogenase.