The dynamical nature of enzymatic catalysis

Abstract

CONSPECTUS: As is well-known, enzymes are proteins designed to accelerate specific life essential chemical reactions by many orders of magnitude. A folded protein is a highly dynamical entity, best described as a hierarchy or ensemble of interconverting conformations on all time scales from femtoseconds to minutes. We are just beginning to learn what role these dynamics play in the mechanism of chemical catalysis by enzymes due to extraordinary difficulties in characterizing the conformational space, that is, the energy landscape, of a folded protein. It seems clear now that their role is crucially important. Here we discuss approaches, based on vibrational spectroscopies of various sorts, that can reveal the energy landscape of an enzyme-substrate (Michaelis) complex and decipher which part of the typically very complicated landscape is relevant to catalysis. Vibrational spectroscopy is quite sensitive to small changes in bond order and bond length, with a resolution of 0.01 Å or less. It is this sensitivity that is crucial to its ability to discern bond reactivity. Using isotope edited IR approaches, we have studied in detail the role of conformational heterogeneity and dynamics in the catalysis of hydride transfer by LDH (lactate dehydrogenase). Upon the binding of substrate, the LDH·substrate system undergoes a search through conformational space to find a range of reactive conformations over the microsecond to millisecond time scale. The ligand is shuttled to the active site via first forming a weakly bound enzyme·ligand complex, probably consisting of several heterogeneous structures. This complex undergoes numerous conformational changes spread throughout the protein that shuttle the enzyme·substrate complex to a range of conformations where the substrate is tightly bound. This ensemble of conformations all have a propensity toward chemistry, but some are much more facile for carrying out chemistry than others. The search for these tightly bound states is clearly directed by the forces that the protein can bring to bear, very much akin to the folding process to form native protein in the first place. In fact, the conformational subspace of reactive conformations of the Michaelis complex can be described as a "collapse" of reactive substates compared with that found in solution, toward a much smaller and much more reactive set. These studies reveal how dynamic disorder in the protein structure can modulate the on-enzyme reactivity. It is very difficult to account for how the dynamical nature of the ground state of the Michaelis complex modulates function by transition state concepts since dynamical disorder is not a starting feature of the theory. We find that dynamical disorder may well play a larger or similar sized role in the measured Gibbs free energy of a reaction compared with the actual energy barrier involved in the chemical event. Our findings are broadly compatible with qualitative concepts of evolutionary adaptation of function such as adaptation to varying thermal environments. Our work suggests a methodology to determine the important dynamics of the Michaelis complex.

Figure 1

LDH catalyzes the interconversion of NADH + pyruvate + H⁺ with NAD⁺ + lactate (see refs ( and 27)). Binding is strictly ordered with cofactor binding preceding substrate. It is widely believed that hydride and proton transfer are concerted. Calculations show multiple routes for proton and hydride transfer occurring in traversal of the transition state within the time frame of a single bond vibration (ca. 5 fs). Shown is a schematic of the LDH active site showing the residues stabilizing the substrate pyruvate and the proximity of the cofactor, NADH. The catalytically key surface loop (residues 98–110) closes over the active site, bringing residue Arg109 in hydrogen bond contact with the ligand; water leaves the pocket. Creation of the pocket is accompanied by the motions of mobile areas within the protein, rearranging the pocket geometry to allow for favorable interactions between the cofactor and the ligand that facilitate on-enzyme catalysis. Of particular interest to this work are the hydrogen bonds formed between Arg109 and His195 to the C2 carbonyl of pyruvate (emphasized in red). These bonds dictate the polarity of the carbonyl when pyruvate is bound. Figure taken from ref (35).

Figure 2

(a) IR spectrum of pyruvate in water. (b) FTIR isotope edited difference spectrum (D168N)LDH·NADH·[¹³C₂]pyruvate subtracted from that of (D168N)LDH·NADH·[¹²C₂]pyruvate. The D168NLDH mutants shows a k_cat about a factor of 800 lower than native LDH. (c) FTIR isotope edited difference spectrum with the spectrum of LDH·NADH·[¹³C₂]pyruvate subtracted from that of LDH·NADH·[¹²C₂]pyruvate. The spectral region of the ¹³C=O stretch is considerably downshifted from ¹²C=O stretch and is not shown in the figure. The difference spectra are measured using [¹³C/¹⁵N]LDH labeled protein to move the intense amide-I protein IR band out of the way of pyruvate’s ¹²C=O stretch. Figure adapted from ref (22).

Scheme 1. Best Fit Kinetic Scheme of the IR Transients in Figure 3

Figure 3

Isotope-labeled IR difference temperature-jump relaxation transients of LDH·NADH·[¹²C₂]pyruvate minus LDH·NADH·[¹³C₂]pyruvate at various probe frequencies. Each probe frequency is plotted as a different color as specified in the legend, and the exponential fits using kinetic Scheme 1 are plotted as black lines. The Michaelis state transients each show a negative amplitude signal with a submillisecond relaxation lifetime that depends on the probe frequency: 254 μs (1685 cm^–1), 128 μs (1679 cm^–1), and 44 μs (1670 cm^–1). The difference transients are measured using [¹³C/¹⁵N]LDH uniformly labeled protein to move the intense amide-I protein IR band out of the way of pyruvate’s ¹²C=O stretch. Graph adapted from ref (35).