Michael G. Vincent

Spatial Aspects of Catalysis by Mitochondrial Aspartate Aminotransferase

Inspection of the structural models of five refined crystal structures of different unliganded and liganded forms of mitochondrial aspartate aminotransferase (McPhalen et al.P this Volume) has led to a number of observations about ligand binding and conformational changes involving both the “small domain” and the coenzyme that are relevant to the catalytic mechanism of this enzyme.

Crystallographic and Solution Studies on Phosphoserine Aminotransferase (PSAT) from E.coli

Phosphoserine aminotransferase from E.coli has been crystallized in space group P212121, with one ∝2 dimeric molecule per asymmetric unit. Single crystal microspectrophotometric measurements have shown that the enzyme is catalytically active in the crystal. X-Ray data for the native form and 2 heavy atom derivatives have been collected on a CAD4-di f fractometer. A 6A resolution Fourier-map has been calculated? interpretation is underway. The orientation of the noncrystallographic two-fold axis of the dimer has been determined. Solution studies have shown that the activity maximum is below the pK of the internal aldimine. The quaternary and secondary structure have been determined by analytical ultracentrifugation and CD measurements.

Recent Studies on Mitochondrial Aspartate Aminotransferase: Structure & Mechanism

The 3-dimensional structures of five forms of the mitochondrial aspartate aminotransferase (mAAT) from chicken heart have been solved by X-ray crystallography. The goal of our work is to understand the catalytic mechanism of AAT by studying the structures of analogs of catalytic intermediates. Comparisons among the structures confirm previous observations of gross conformational changes during catalysis. More subtle differences are seen in several regions of the protein, including the active site, based on the refined higher-resolution structures.

Phosphorus-31 Nuclear Magnetic Resonance Studies on Apoaspartate Aminotransferase Reconstituted with Cofactor Analogues

The 31P chemical shift of pyridoxal phosphate (PLP) in native cytosolic aspartate aminotransferase (cAAT) has been reported to be pH dependent with a pK of 6.2 (Schnackerz, 1984) even though X-ray data suggest that the cofactor phosphate group remains dianionic throughout. For further information on the pH dependence of 31P NMR spectra apo-cAAT reconstituted with phosphopyridoxyl aspartate and pyridoxal 5′deoxymethylene-phosphonate was measured. The chemical shifts for the two cofactor analogues when bound to apo-cAAT were found to be pH independent and should correspond to the phosph(on)ate dianion. Thus, the 31P NMR data on native cAAT can only be interpreted as the protonation/deprotonation equilibrium of the PLP-Lys 258 “internal aldimine” (pK=6.2). It is proposed that protonation of the aldimine exerts strain in the 5′-phosphate ester linkage, resulting in modified O-P-O bond angles which in turn cause changes in the 31P chemical shift.

Glutamate Decarboxylase: Preliminary Crystallographic Data

Glutamate decarboxylase was purified from Escherichia coli and crystallized by vapour diffusion and microdialysis techniques in the presence of polyethylene glycol. Growth of large crystals suitable for X-ray studies is particularly favoured by low concentrations of citric or glutaric acid, which are both effective inhibitors of the enzyme. Inhibitor binding must induce the conformational changes that are essential for crystal nucleation since no crystallization occurred with non-inhibitory buffers of similar ionic composition. The crystals, of space group R3, diffract to a resolution of 2.9 Ȧ. The dimensions of the rhombohedral unit cell are a = b = c = 116 Ȧ, α= β = γ =116° with a dimer in the asymmetric unit. The equivalent hexagonal cell has a = b = 199 Ȧ, c = 69 Ȧ, α= β = 90°, γ= 120°.