Aspartate aminotransferase: An old dog teaches new tricks

Abstract

Aspartate aminotransferase (AAT) is a prototypical pyridoxal 5′-phosphate (PLP) dependent enzyme that catalyzes the reversible interconversion of L-aspartate and α-ketoglutarate with oxalacetate and L-glutamate via a ping-pong catalytic cycle in which the pyridoxamine 5′-phosphate enzyme form is an intermediate. There is a bountiful literature on AAT that spans approximately 60 years, and much fundamental mechanistic information on PLP dependent reactions has been gained from its study. Here, we review our recent work on AAT, where we again used it as a test bed for fundamental concepts in PLP chemistry. First, we discuss the role that coenzyme protonation state plays in controlling reaction specificity, then ground state destabilization via hyperconjugation in the external aldimine intermediate is examined. The third topic is light enhancement of catalysis of Cα-H deprotonation by PLP in solution and in AAT, which occurs through a triplet state of the external aldimine intermediate. Lastly, we consider recent advances in our analyses of enzyme multiple sequence alignments for the purpose of predicting mutations that are required to interconvert structurally similar but catalytically distinct enzymes, and the application of our program JANUS to the conversion of AAT into tyrosine aminotransferase.

Expression, purification and preliminary crystallographic studies of human glutamate oxaloacetate transaminase 1 (GOT1)

“…GOT with pyridoxal 5 0 -phosphate (PLP) as an essential cofactor [12]. Cellular microorganisms contain only a single GOT, whereas two genetically distinct isoenzymes have been found in eukaryotic cells, one located in the cytoplasm, and the other in mitochondria.…”

Phylobiochemical Characterization of Class-Ib Aspartate/Prephenate Aminotransferases Reveals Evolution of the Plant Arogenate Phenylalanine Pathway

“…Aminotransferases catalyze a PLP-dependent transamination reaction between an a-amino acid donor and an a-keto acid amino acceptor via the "ping-pong bi-bi" mechanism (Kirsch et al, 1984;Toney, 2014). The AspAT Ib enzymes were previously shown to preferentially use dicarboxylic acid amino donors (i.e., aspartate and glutamate), in contrast to class Ia AspATs that use both acidic and neutral amino donors (Kuramitsu et al, 1990;Nakai et al, 1999).…”

Supplementation of metabolizable protein during late gestation and fetal number impact ewe organ mass, maternal serum hormone and metabolite concentrations, and conceptus measurements

“…In ruminants, urea entering the rumen is degraded to ammonia, then used to synthesize amino acids and other nitrogen-containing compounds, or absorbed into venous drainage [21]. Aspartate is synthesized by a 1-step transamination reaction catalyzed by AST using oxaloacetate as the precursor [22]. Aspartate is a precursor of ornithine in the urea cycle [23].…”