Decreased Transition-State Analogue Affinity in Isotopically Heavy MTAN with Increased Catalysis

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 (K_i) and for the tightly bound complex after slow-onset inhibition (K_i*) with hexylthio-DADMe-Immucillin-A (HTDIA) gave K_i values for light and heavy HpMTAN = 52 ± 10 and 85 ± 13 pM and K_i* 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.