Aspartate aminotransferase active sites

Beta-chloroalanine and serine O-sulfate can undergo (3 elimination (as in Eq. 14-29) in active sites of glutamate decarboxylase or aspartate aminotransferase. The enzymes then form free aminoacrylate, a reactive molecule that can undergo an aldol-type condensation with the external aldimine to give the following product.1... 

Figure 14-6 Drawing showing pyridoxal phosphate (shaded) and some surrounding protein structure in the active site of cytosolic aspartate aminotransferase. This is the low pH form of the enzyme with an N-protonated Schiff base linkage of lysine 258 to the PLP. The tryptophan 140 ring lies in front of the coenzyme. Several protons, labeled Ha, Hb, and Hd are represented in NMR spectra by distinct resonances whose chemical shifts are sensitive to changes in the active site.169...

Figure 14-10 Models of catalytic intermediates for aspartate aminotransferase in a half-transamination reaction from aspartate to oxalocetate. For clarity, only a selection of the active site groups are shown. (A) Michaelis complex of PLP enzyme with aspartate. (B) Geminal diamine. (C) Ketimine intermediate. The circle indicates a bound water molecule. See Jansonius and Vincent in Jurnak and McPherson.163 Courtesy of J.N. Jansonius.

Below the active site of aspartate aminotransferase, as shown in Fig. 14-6, is a cluster of three buried histidine side chains in close contact with each other. The imidazole of H143 is hydrogen bonded to the D222 carboxylate, the same carboxylate that forms an ion pair with the coenzyme. This system looks somewhat like the catalytic triad of the serine proteases in reverse. As with the serine proteases, the proton-labeled Hb in Fig. 14-6 can be "seen" by NMR spectroscopy (Fig. 3-30). So can the proton Ha on the PLP ring. These protons... 

Aspartate aminotransferase 57s, 135s, 753 absorption spectra 749 active site structure 744 atomic structure 750 catalytic intermediates, models 752 NMR spectra 149 quinonoid intermediate 750 Ramachandran plot 61 sequence 57 transamination 742 Aspartate ammonia-lyase 685 Aspartate carbamoyltransferase 348s active sites 348 regulation 540... 

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.

Branched-chain aminotransferases (BCATs) evolved from aspartate aminotransferases (AATs) showed a record 105- to 2 x 106-fold improvement in catalytic efficiency (kcat/KM). Not only were the 13-17 amino acid substitutions concentrated in the most active mutants, but all but one mutated amino acid residues are located far from the active site. With directed evolution, enantioselectivities can be improved on enantiounspecific enzymes (from E = 1.1 to 25.8) and even inverted to yield the opposite enantiomer in comparison to the wild type (40% d- to both 90% d- and 20% L-). 

P. Strop, H. Gehring, J. N. Jansonius, and P. Christen, Conversion of aspartate aminotransferase into an L-aspartate /3-decarboxylase by a triple active-site mutation, J. Biol. Chem. 1999, 274, 31203-31208. 

Active site structure of Escherichia coli aspartate aminotransferase (see Chapter 5 for details). 

Many of the enzymes that catalyze these reactions, such as serine hy- droxymethyltransferase, which converts serine into glycine, have the same fold as that of aspartate aminotransferase and are clearly related by divergent evolution. Others, such as tryptophan synthetase, have quite different overall structures. Nonetheless, the active sites of these enzymes are remarkably similar to that of aspartate aminotransferase, revealing the effects of convergent evolution. 

Figure 23.11. Aspartate Aminotransferase. The active site of this prototypical PLP-dependent enzyme includes...

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. 

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