Steven Dajnowicz(1,2), Timothy C. Mueser(1), Andrey Kovalevsky(2)
(1) University of Toledo Department of Chemistry, (2) Oak Ridge National Laboratory Neutron Scattering Division
Enzymes dependent on pyridoxal 5’-phosphate (PLP) perform diverse and complex organic reactions, affording wide spread involvement in cellular metabolism. To promote reaction diversity and catalysis, PLP-dependent enzymes exploit the electronic resonance prosperities of the vitamin B6 derived cofactor. Moreover, the two major physical determinants that govern reaction specificity are 1) stereoelectronic control of bond cleavage and 2) electronic modulation through selective protonation of the cofactor, induced by the active site local environment. Our current effort is to understand the physical phenomena that drives reaction specificity and catalysis, allowing future developments for medicine and protein engineering purposes.
Neutron crystallography has the unique ability of visualizing the nuclear positions of the reactive hydrogen atoms in enzymes. Here we present a room temperature neutron structure of a homodimeric PLP-dependent enzyme, aspartate aminotransferase (AAT). Moreover, the neutron structure of AAT contains both the internal aldimine (PLP covalently linked to the enzymes) and external aldimine (PLP covalently linked to the substrate) forms of the enzyme within one crystal. The AAT neutron structure provides direct evidences of different active site protonation profiles between the internal and external aldimine forms of the enzymes. Specially, in the external aldimine state of AAT, a low-barrier hydrogen bond is observed between the Schiff base nitrogen and C-terminal carboxylate of the substrate, which maybe critical for catalysis. In addition, the neutron structure was utilized as an input for molecular simulations, providing evidence that hyperconjugation causes a 1012 rate enhancement for the transamination reaction in AAT.