Ira E. Smiley

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.

Single crystal x-ray diffraction studies of Southern bean mosaic virus.

Crystals of the cowpea strain of Southern bean mosaic virus were shown to belong to space group R32 whose hexagonal cell dimensions were a = 923(1), c = 299(1) . The orientation and position of the three virus particles in the rhombohedral cell were determined by a consideration of intensity spikes, packing arrangements, and the relative size of different classes of Bragg reflections. The unusually open packing arrangement was subsequently confirmed by electron microscopy observation of thin-sectioned crystals. A maximum particle diameter was found to be 284 . The X-ray diffraction patterns extend to 3 resolution.

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.

Subunit Interactions in Lactate Dehydrogenase

The NAD+ dependent tetrameric enzyme lactate dehydrogenase (EC 1.1.1.27), of molecular weight 140,000 Daltons, catalyzes the interconversion of L (+) lactate and pyruvate. Five isoenzymes result from the two subunit types abundant in tissues. The H subunit type is predominant in heart tissue and the M type in skeletal muscle. The M4 isoenzyme (LDH-5) from dogfish (squalus acanthius) has been most studied in this laboratory, while investigations are now proceeding on the H4 (LDH-1) and M4 (LDH-5) isoenzyme of pig.

Crystalline cowpea chlorotic mottle virus

A variety of crystal forms of cowpea chlorotic mottle virus were grown, one of which gave useful X-ray diffraction diagrams. The unit cell has cubic symmetry of space group F432. The diameter of the virus was found to be consistent with that determined from electron microscopy studies.

Structure and Mechanism of Lactate Dehydrogenase

The enzyme lactate dehydrogenase (LDH) catalyses the conversion of lactate to pyruvate in the presence of the coenzyme nicotinamide adenine dinucleotide (NAD). It is composed of four subunits each of molecular weight 35,000 (Appella and Markert, 1961; Cahn et al, 1962). The subunits are single polypeptide chains of either H (heart) or M (muscle) variety (Wieland and Pfleiderer, 1957; Markert and Moller, 1959). Hybridization of these chains gives rise to five possible isoenzymes per species. This paper concerns itself with the properties of the M4 isoenzyme of dogfish (Squalus acanthius). Rossmann et al (1967) have previously reported that the apo-enzyme crystallizes in space group F422 with a = 146.9, c = 155.2A, and one polypeptide chain per asymmetric unit. Adams et al (1969) showed that the molecular center coincided with the intersection of a defined set of mutually perpendicular two-fold axes in the crystal lattice. They also reported a low resolution (5.0A) structure in which the boundaries of the molecule and subunits could be traced. In addition some properties of the binary coenzyme complex were discussed.