Enzymatic Transition States, Transition-State Analogs, Dynamics, Thermodynamics, and Lifetimes

Abstract

Experimental analysis of enzymatic transition-state structures uses kinetic isotope effects (KIEs) to report on bonding and geometry differences between reactants and the transition state. Computational correlation of experimental values with chemical models permits three-dimensional geometric and electrostatic assignment of transition states formed at enzymatic catalytic sites. The combination of experimental and computational access to transition-state information permits (a) the design of transition-state analogs as powerful enzymatic inhibitors, (b) exploration of protein features linked to transition-state structure, (c) analysis of ensemble atomic motions involved in achieving the transition state, (d) transition-state lifetimes, and (e) separation of ground-state (Michaelis complexes) from transition-state effects. Transition-state analogs with picomolar dissociation constants have been achieved for several enzymatic targets. Transition states of closely related isozymes indicate that the protein’s dynamic architecture is linked to transition-state structure. Fast dynamic motions in catalytic sites are linked to transition-state generation. Enzymatic transition states have lifetimes of femtoseconds, the lifetime of bond vibrations. Binding isotope effects (BIEs) reveal relative reactant and transition-state analog binding distortion for comparison with actual transition states.

Femtosecond dynamics coupled to chemical barrier crossing in a Born-Oppenheimer enzyme

“…Intrinsic KIEs for labeled inosines as substrates for PNP reflect differences in bond vibrational environment between substrates in solution and at the transition state (24). Accordingly, KIE analysis has been used to probe transition-state structures of PNP as well as several related enzyme-catalyzed reactions (6,(27)(28)(29)(30). The intrinsic KIEs of heavy and light PNP are indistinguishable within experimental error ( Table 1), indicating that altered femtosecond protein dynamic motions do not change the geometry of the PNP transition state.…”

“…ES ¼ ðE × SÞ∕ðS þ K M Þ: [6] Saturation Curves Under Steady-State Conditions. Apparent steady-state parameters were determined by measuring initial rates as a function of either guanosine or inosine concentration at 25°C.…”

“…The role of protein dynamics has been brought into question with the suggestion that electrostatic preorganization is the sole force responsible for the catalytic prowess of enzymes (3). However, the idea that dynamics is involved in enzymatic catalysis is more widely accepted and is supported by theoretical and experimental work (4)(5)(6). The temporal similarity of catalysis and slow conformational changes has led to proposals that chemistry (crossing the transition-state barrier) is linked to millisecond motions of proteins (1,7).…”

Transition states of native and drug-resistant HIV-1 protease are the same

“…This method for elucidating enzymatic transition structures has provided sufficiently robust transition-state knowledge to permit design and synthesis of powerful transition-state mimics (34). Here, we apply transition-state analysis to the study of the chemical mechanism of HIV-1 protease.…”

Structural Snapshots for Mechanism‐Based Inactivation of a Glycoside Hydrolase by Cyclopropyl Carbasugars

“…Given that enzyme dynamics is an important component to catalysis,5b our findings provide a basis for designing TSA inhibitors that incorporate the features of the bicyclobutenium ion intermediate resulting from the reaction of these novel bicyclo[4.1.0]heptane inactivators.…”