Differences in the binding of coenzyme to L-3-hydroxyacyl-coenzyme A dehydrogenase in the crystalline state and in solution.

L-3-Hydroxyacyl coenzyme A dehydrogenase (L-3hydroxyacyl-CoA:NAD oxidoreductase; EC 1 .l .1.35) is a mitochondrial enzyme involved in the beta oxidation of fatty acids. The enzyme, as isolated from pig heart muscle, hasM, 67 000 and contains two identical subunits [l]. The amino acid sequence has recently been completed and appears to have little homology with other dehydrogenases [2]. Like many other dehydrogenases, L-3-hydroxyacyl coenzyme A dehydrogenase @-HADH) utilizes NAD-NADH as cofactors. However, unlike many other dehydrogenases, the structure of its substrate, L-3-hydroxyacyl coenzyme A, resembles the cofactor, NAD. Structural studies of the enzyme should prove to be of interest in that both the substrate and coenzyme binding sites might be expected to contain binding domains for adenine nucleotide moieties. In addition, the active site of /3-HADH must accommodate the varying acyl chain lengths of fatty acids that are metabolized through the beta oxidation cycle. In order to determine the molecular structure of P-HADH, an X-ray crystallographic study is being carried out. From an electron density map at low resolution, the tentative location of the two dimers contained in the asymmetric unit has been determined [3]. In addition, crystallographic studies aimed at locating the binding site of NAD have also been undertaken. Difference Fourier maps between the holoand apo-forms of the crystalline enzyme yield only 1 major NAD binding site/dimer. This apparent asymmetry in NAD binding to crystalline /3-HADH prompted a study of the binding characteristics of coenzyme to fl-HADH in solution. This communication reports the comparison of the X-ray and solution studies. Pig heart L3-hydroxyacyl-CoA dehydrogenase was prepared as in [4] and crystals suitable for X-ray diffraction studies were grown in the presence of polyethylene glycol-6000 [5]. For solution studies, the crystals were centrifuged, the solvent decanted, and the crystalline precipitate dissolved in 50 mM phosphate buffer (pH 7.0). The enzyme was then passed through a 0.7 X 20 cm Sephadex G-25 column to insure complete removal of the crystallizing medium. The enzyme concentration was determined using a E$$ = 4.39 [6] and subunit Mr 33 500 [2]. NADH (grade III) was obtained from Sigma Chemical Co. (St Louis MO) and was used without further purification. All other chemicals were reagent grade. Binding measurements were determined by examining changes in the intrinsic protein fluorescence upon binding of NADH. Fluorescence measurements were made on a Spex Fluorolog spectrofluorimeter using an excitation wavelength of 290 nm (bandpass of 3 nm) and observing the emission at 350 nm (bandpass of 40 nm). All measurements were performed at 20°C in a 50 mM potassium phosphate buffer (pH 7.0) which was 1 mM with respect to both EDTA and 2-mercaptoethanol. The enzyme was titrated by the addition of 4 ~1 quantities of titrant to a constantly stirred 3 .O ml contained in a 1 .O X 1 .O cm quartz cuvette. Fluorescence corrections were made by an identical titration of a buffer blank. These values were subtracted from the sample fluorescence. The concentration of free NADH was calculated from the following relationship: