%0 Journal Article
%T Decreased Transition-State Analogue Affinity in Isotopically Heavy MTAN with Increased Catalysis.
%A Brown M
%A Schramm VL
%J Biochemistry
%V 62
%N 20
%D 2023 10 17
%M 37788145
%F 3.321
%R 10.1021/acs.biochem.3c00434
%X 5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase from Helicobacter pylori (HpMTAN) demonstrated faster chemistry when expressed as an isotopically heavy protein, with 2H, 13C, and 15N replacing the bulk of normal isotopes. The inverse heavy enzyme isotope effect has been attributed to improved enzyme-reactant interactions causing more frequent transition-state formation ( Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2109118118). Transition-state analogues stabilize the transient dynamic geometry of the transition state and inform on transition-state dynamics. Here, a slow-onset, tight-binding transition-state analogue of HpMTAN is characterized with heavy and light enzymes. Dissociation constants for the initial encounter complex (Ki) and for the tightly bound complex after slow-onset inhibition (Ki*) with hexylthio-DADMe-Immucillin-A (HTDIA) gave Ki values for light and heavy HpMTAN = 52 ± 10 and 85 ± 13 pM and Ki* values = 5.9 ± 0.3 and 10.0 ± 1.2 pM, respectively. HTDIA dissociates from heavy HpMTAN at 0.063 ± 0.002 min-1, faster than that from light HpMTAN at 0.032 ± 0.004 min-1. These values are consistent with transition-state formation by an improved catalytic site dynamic search and inconsistent with catalytic efficiency proportional to tight binding of the transition state.