Transition States and transition state analogue interactions with enzymes

Abstract

Enzymatic transition states have lifetimes of a few femtoseconds (fs). Computational analysis of enzyme motions leading to transition state formation suggests that local catalytic site motions on the fs time scale provide the mechanism to locate transition states. An experimental test of protein fs motion and its relation to transition state formation can be provided by isotopically heavy proteins. Heavy enzymes have predictable mass-altered bond vibration states without altered electrostatic properties, according to the Born-Oppenheimer approximation. On-enzyme chemistry is slowed in most heavy proteins, consistent with altered protein bond frequencies slowing the search for the transition state. In other heavy enzymes, structural changes involved in reactant binding and release are also influenced. Slow protein motions associated with substrate binding and catalytic site preorganization are essential to allow the subsequent fs motions to locate the transition state and to facilitate the efficient release of products. In the catalytically competent geometry, local groups move in stochastic atomic motion on the fs time scale, within transition state-accessible conformations created by slower protein motions. The fs time scale for the transition state motions does not permit thermodynamic equilibrium between the transition state and stable enzyme states. Isotopically heavy enzymes provide a diagnostic tool for fast coupled protein motions to transition state formation and mass-dependent conformational changes. The binding of transition state analogue inhibitors is the opposite in catalytic time scale to formation of the transition state but is related by similar geometries of the enzyme-transition state and enzyme-inhibitor interactions. While enzymatic transition states have lifetimes as short as 10(-15) s, transition state analogues can bind tightly to enzymes with release rates greater than 10(3) s. Tight-binding transition state analogues stabilize the rare but evolved enzymatic geometry to form the transition state. Evolution to efficient catalysis optimized this geometry and its stabilization by a transition state mimic results in tight binding. Release rates of transition state analogues are orders of magnitude slower than product release in normal catalytic function. During catalysis, product release is facilitated by altered chemistry. Compared to the weak associations found in Michaelis complexes, transition state analogues involve strong interactions related to those in the transition state. Optimum binding of transition state analogues occurs when the complex retains the system motions intrinsic to transition state formation. Conserved dynamic motion retains the entropic components of inhibitor complexes, improving the thermodynamics of analogue binding.

Figure 1

Time scales for motions related to enzyme catalysis arranged in orders of magnitude. The top line represents 1000 s, and the bottom line represents 0.000000000000001 s.

Figure 2

Human PNP catalytic site filled with phosphate and DADMe Immucillin-G (upper panel). The phosphate and DADMe Immucillin-G ligands are in orange. The PNP-phosphate structure (yellow shading) shows His64 (loop 2) and His257 (loop 1) turned out of the catalytic site. The PNP structure with the catalytic site filled with phosphate and DADMe Immucillin-G, a 2 pM transition state analogue inhibitor (green shading), shows His64 and His257 turned in to make hydrogen bond contacts. The green arrows indicate the His relocation motions. Only six of more than a dozen interactions are shown. Ribbons represent α-helical segments, and tubes represent loop structures. The lower panel shows transition state analogues of human PNP.

Figure 3

Catalytic site of human PNP with Immucillin-H (ImmH) and inorganic phosphate (PO₄) at the catalytic site. The PO₄ is buried in the protein. Side chains from Y88, F159, F200, and H257 form a layer between the catalytic site and solvent. F159 is from the adjacent subunit (dark blue). It must relocate to permit product release (blue arrow), the rate-limiting step (RLS) of catalysis.

Figure 4

Motion of His257 with inosine at the catalytic site of in human PNP and transition state formation. The upper panel shows three of the amino acid contacts to reactants in the Michaelis complex. His257 forms a hydrogen bond to the O5′ hydroxyl group and orients the 5′ oxygen toward the 4′-ribose ring oxygen. The interaction (pink curved arrow) is transient, on the ns time scale, as indicated in molecular mechanics calculation (middle panel). (Reprinted with permission from ref . Copyright 2010 American Chemical Society.) The three dynamic traces show the interactions in each of the three catalytic subunits of PNP (green, red, and black) on the ns time scale. The His257 interaction causes compression of the O4′–O5′ distance, destabilizing the N-ribosidic bond. Minimization of the O4′–O5′ distance is associated with transition state formation (lower panel; copyright 2008, Proceedings of the National Academy of Sciences, U.S.A.). The transition state has a lifetime of 10 fs (yellow bar, lower panel) as determined by unbiased quantum chemical transition path sampling. The local dynamic search for the transition state for this interaction has a oscillation period of approximately 30 fs. In the 5 ns period where the His257N–O5′ hydroxyl interaction is maintained, the 30 fs search can occur 167 000 times. Time scales in the middle and lower panels differ by 40 000.

Figure 5

Chemical consequences of introducing solvent access to the catalytic sites of PNP. Refer to Figure 3 for the location of these amino acids. The normal reaction and transition state are shown in the upper reaction path. Catalytic site mutations do not permit formation of ribose (lower right), but it does form if phosphate is absent. Mutations F200G and H257G permit misalignment of the transition state reactants and N3-isoinosine forms (lower left).

Figure 6

Cartoons of the distinct time scales for PNP catalysis and transition state analogue binding. Substrate and the pretransition state conformational changes are on the ms to ns time scale (upper panel). Within these times, the local catalytic site contacts (red dashed lines) vary their distance on the fs time scale. Optimized fs interactions locate the transition state which exists only for 10 fs. The fs excursions are from a relaxed form of the enzyme. When a transition state analogue is bound (bottom panel), each of the interactions is stabilized (red arrows), the protein condenses around the analogue, and the motions for release become orders of magnitude slower than in the normal catalytic cycle.