Aspartate aminotransferase subunits

Figure 3-23 (A) Stereoscopic a-carbon plot of the cystolic aspartate aminotransferase dimer viewed down its dyad symmetry axis. Bold lines are used for one subunit (subunit 1) and dashed lines for subunit 2. The coenzyme pyridoxal 5 -phosphate (Fig. 3-24) is seen most clearly in subunit 2 (center left). (B) Thirteen sections, spaced 0.1 nm apart, of the 2-methylaspartate difference electron density map superimposed on the a-carbon plot shown in (A). The map is contoured in increments of 2a (the zero level omitted), where a = root mean square density of the entire difference map. Positive difference density is shown as solid contours and negative difference density as dashed contours. The alternating series of negative and positive difference density features in the small domain of subunit 1 (lower right) show that the binding of L-2-methylaspartate between the two domains of this subunit induces a right-to-left movement of the small domain. (Continues)...

Figure 23-9 Polarized absorption spectra of orthorhombic crystals of cytosolic aspartate aminotransferase. The light beam passed through the crystals along the b axis with the plane of polarization parallel to the a axis (A) or the c axis (C). Left, native enzyme at pH 5.4 right, enzyme soaked with 300 mM 2-methylaspartate at pH 5.9. The band at 430 nm represents the low pH proto-nated Schiff base form of the enzyme. Upon soaking with 2-methylaspartate the coenzyme rotates 30° to form a Schiff base with this quasisubstrate. The result is a large change in the c/a polarization ratio. The 364 nm band in the complex represents the free enzyme active site in the second subunit of the dimeric enzyme.70,73 Courtesy of C. M. Metzler.

The a subunit catalyzes the formation of indole from indole-3-glycerol phosphate, whereas each P subunit has a PLP-containing active site that catalyzes the condensation of indole and serine to form tryptophan. The overall three-dimensional structure of this enzyme is distinct from that of aspartate aminotransferase and the other PLP enzymes already discussed. Serine forms a Schiff base with this PLP, which is then dehydrated to give the Schiffbase of aminoacrylate. This reactive intermediate is attacked by indole to give tryptophan. 

Figure 23.12 Aspartate aminotransferase. The active site of this prototypical PLP-dependent enzyme includes pyridoxal phosphate attached to the enzyme by a Schiff-base linkage with lysine 2S8, An arginine residue in the active site helps orient substrates by binding to their u-carboxylate groups. Only one of the enzyme s two subunits is shown,...

Aspartate aminotransferase (AAT) is the first PLP-dependent enzyme for which the three-dimensional structure has been determined " " and is the prototype of fold-type I PLP-enzymes. Each subunit of the AAT homodimer has a large and a small domain. The coenzyme is bound to the large (N-terminal) domain and located in a pocket at the subunit interface, so that residues from each monomer contribute to the formation of both active sites. The proximal and distal carboxylate group of the dicarboxylic substrates bind to Arg386 and Arg292, respectively, the latter contributed by the opposite subunit. " Early crystallographic strucmres... 

The well-characterized cytosolic and mitochondrial aspartate aminotransferases of pig heart are dimeric, comprising two subunits of essentially identical size. Studies of cofactor content and primary structure, plus demonstrations of physical dissociation into constituent subunits, all confirm the dimeric structure of the pig enzyme (cf. Braunstein, 1973). Comparable evi-... 

FIGURE 8 Structure of an aspartate aminotransferase. The protein is a homodimer, with one covalently bound pyridoxal phosphate (shown in black) in each of the two subunits. The expanded view shows the cofactor in greater detail. [Adapted from Rhee, S. et al. (1997). Refinement and comparisons of the crystal structures of pig cytosolic aspartate aminotransferase and its complex with 2-methylaspartate, J. Biol. Chem. 272,17293-17302.]... 

Model studies (pyridoxal catalyzed conversion of a-amino acid to oxo-acid) indicates that the prototropic shift is in the aldimine <—> ketimine tautomerization, and this step can be greatly accelerated by general acid-base catalysis. Aspartate ( 2-oxoglutarate) aminotransferase (EC 2.6.1.1), which catalyzes transamination between Asp and 2-oxoglutarate (oxaoacetate and Glu), is the most extensively studied representative PLP enzyme. The enzyme is a homodimer containing one PLP molecule per subunit. Experimental observations pertaining to apartate aminotransferase are ... 

---