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 wild-type 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 When Asn(194) had been replaced by Ala in advance, the V39F mutation did not decrease the aldimine pK(a), showing that the domain rotation controls the aldimine pK(a) via the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(38) carbonyl O and Tyr(225) Oeta seemed to be the cause of the reduced pK(a) value. Kinetic analysis suggested that the repulsion increases the free energy level of the Michaelis complex and promotes the catalytic reaction.
Highlights
The atomic coordinates and structure factors have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
Because the aldimine pKa value is closely related to the presence of the hydrogen bond between pyridoxal 5Ј-phosphate (PLP) O3Ј and Asn194 [14, 15], the effect of the V39F mutation was studied on the N194A mutant AspAT (Fig. 1)
In this study on AspAT, we observed that the conformational change in the enzyme protein significantly affects the pKa of the PLPLys258 aldimine at the active site
Summary
AspAT, aspartate aminotransferase (aspartate, 2-oxoglutarate aminotransferase, EC 2.6.1.1); PLP, pyridoxal 5Ј-phosphate; PMP, pyridoxamine 5Ј-phosphate; V39F AspAT, mutant AspAT in which the residue Val has been replaced with a phenylalanine residue (other mutant AspATs are expressed in the same way); WT, wild-type; MES, 4-morpholineethanesulfonic acid; TAPS, 3-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}-1-propanesulfonic acid. The increase in the aldimine pKa in the Michaelis complex has been claimed to accelerate the transaldimination step (ELHϩ1⁄7S 3 ELHϩϭS in Scheme I) by shifting the equilibrium from EL1⁄7SHϩ to ELHϩ1⁄7S in the Michaelis complex [10], its exact role in catalysis is obscure, because the transaldimination step is not rate-determining at all in the catalytic reaction [16] To solve these problems associated with the Michaelis complex, we set out to carry out structural and mechanical analysis of the complex. We have chosen the mutation of Val39 [19], which exists at the interface of the large and small domains, to a more bulky residue Phe, with the anticipation of hampering the domain rotation and side-chain packing This mutant enzyme was analyzed together with the wild-type enzyme, to explore the effect of the substrate-induced conformational change on the important catalytic groups
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
