Conformational Change in Aspartate Aminotransferase on Substrate Binding Induces Strain in the Catalytic Group and Enhances Catalysis

Abstract

Aspartate aminotransferase has been known to undergo a significant conformational change, in which the small domain approaches the large domain, and the residues at the entrance of the active site pack together, on binding of substrates. Accompanying this conformational change is a two-unit increase in the pK a of the pyridoxal 5-phosphate-Lys 258 aldimine, which has been proposed to enhance catalysis. To elucidate how the conformational change is coupled to the shift in the aldimine pK a and how these changes are involved in catalysis, we analyzed structurally and kinetically an enzyme in which Val 39 located at both the domain interface and the entrance of the active site was replaced with a bulkier residue, Phe. The V39F mutant enzyme showed a more open conformation, and the aldimine pK a was lowered by 0.7 unit compared with the wildtype enzyme. When Asn 194 had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK a , showing that the domain rotation controls the aldimine pK a via the Arg 386 -Asn 194 -pyridoxal 5-phosphate linkage system. The maleate-bound V39F enzyme showed the aldimine pK a 0.9 unit lower than that of the maleate-bound wild-type enzyme. However, the positions of maleate, Asn 194 , and Arg 386 were superimposable between the mutant and the wild-type enzymes; therefore, the domain rotation was not the cause of the lowered aldimine pK a value. The maleate-bound V39F enzyme showed an altered side-chain packing pattern in the 37-39 region, and the lack of repulsion between Gly The homodimeric structures have been solved for cytosolic (2), mitochondrial (3-5), and Escherichia coli (6, 7) enzymes. Each subunit is composed of large and small domains, and the active site containing the PLP-Lys 258 aldimine is located between the two domains. The ␣-carboxylate group of the dicarboxylic substrates binds to Arg 386 located at the small domain and the -carboxylate group to Arg 292 * located at the large domain. An asterisk indicates that the residue comes from the neighboring subunit. On the basis of structural, steady-state (1), and transient (8, 9) kinetic studies, the reaction mechanism of AspAT has been proposed (10, 11) and refined (9) (see Scheme I).The PLP-Lys 258 aldimine (internal aldimine) of the unliganded AspAT has an imine pK a value of ϳ6.5, which is strikingly lower than those of the PLP-amine aldimines with protonated pyridine N (ϳ10; see Ref 13). We have shown that the principle factor that decreases the aldimine pK a value of AspAT is the imine-pyridine torsion of the PLP-Lys 258 aldimine (as represented by the torsion angle C3-C4 -C4Ј-N, expressed as , in the panel of Scheme I), rather than the historically accepted electrostatic effect of the neighboring positive charges of Arg 292 * and Arg 386 . The torsion of the aldimine, the angle of which spans the range from ϳ35°(protonated aldimine) (2) to ϳ90°(unprotonated aldimine) (7), causes a decrease of 3 units in the aldimine pK a (14), whereas the positive charges of the arginine residues decreases the...

Features and technical applications of ω-transaminases

“…Extensive investigations on the biochemical properties of transaminases have provided detailed understanding of the reaction mechanism, especially with aspartate aminotransferase (AspAT) and aromatic amino acid aminotransferase (AroAT) (Hayashi et al 2003;Kagamiyama and Hayashi 2001;Hirotsu et al 2005;Kawaguchi et al 1997;Islam et al 2000;Malashkevich et al 1995;Okamoto et al 1999). 5-TA catalyzes transfer of an amino group from a primary amine to a carbonyl compound mediated by PLP (Shin and Kim 2002).…”

Crystal Structure of Citrobacter freundii Asp214Ala Tyrosine Phenollyase Reveals that Asp214 is Critical for Maintaining a Strain in the Internal Aldimine

“…It was shown that strain in the internal aldimine, caused by the torsion between the PLP pyridine ring and the imine C4′=Nζ bond (Figure 2), plays a considerable role in the transaldimination reaction catalyzed by aspartate aminotransferase. 2,3 Similar strain in the internal aldimine was also found in a number of structures of other PLPdependent enzymes. 2 Moreover, it was demonstrated that exposure to relatively high doses of X-ray radiation relieves the strain in the internal aldimine and even induces breakage of the C4′=Nζ bond in phosphoserine aminotransferase.…”

Transaminases and their Applications

“…Due to structure determination of the aspartate aminotransferase, the reaction mech anism of transaminases is well understood and examined in detail [10,11,[18][19][20]. The transfer of the amino group is supported by the external cofactor PLP forming a Schiff base with the ε-amino group of the lysine on the active side of the enzyme (Lys-E), called internal aldimine.…”

“…More precisely, the internal aldimine undergoes transamination with the amino group of the substrate to create the external aldimine. But it is important that either the internal aldimine is protonated and the amino group is not or the internal aldimine is deprotonated and the amino group is protonated for the extra proton that can be transferred between the amino group of the substrate and the imine nitrogen of the aldimine [19]. The Michaelis-Menten complex formed possesses the proton on the imine nitrogen and the free amino group of the substrate.…”