The mechanism of rate-limiting motions in enzyme function

Abstract

The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, kcat decreases for this mutant and the kinetic solvent isotope effect on kcat, which was 2.0 in WT, is near unity in H48A. Despite being located 18 Å from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of Ϸ160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.Carr-Purcell-Meiboom-Gill dispersion ͉ enzyme dynamics ͉ NMR ͉ protein motions ͉ RNase A C onformational motions in enzymes play an essential role in their function and are often the rate-limiting step to overall catalytic throughput (1-5). Many enzymes have sufficiently evolved such that the bond-making and -breaking steps are fast relative to the ability of the enzyme to undergo a conformational change, and, thus, steps other than the chemical transformation of substrate are rate-limiting (ref. 6 and references therein). In systems such as these, understanding enzyme function requires characterization of the relevant time-dependent protein fluctuations from the timeaveraged three-dimensional structure. The ability of solution NMR spectroscopy to detect, with atomic resolution, motions over a wide timescale (picoseconds to seconds) makes it an ideal experimental technique to characterize conformational motions in proteins that can ultimately impact drug design, de novo enzyme construction, and enzyme engineering. In particular, relaxation-compensated Carr-Purcell-Meiboom-Gill (rcCPMG) dispersion experiments (7) are capable of informing on the kinetics, thermodynamics, and structural changes of protein motions in the microsecond-tomillisecond timescale.RNase A is an enzyme example in which a conformational change is the bottleneck to overall conversion of substrate to product (see ref. 3 for a review). RNase A catalyzes the cleavage of single-stranded RNA and does not require metal ions or cofactors. This enzyme has been studied in great detail as a model for protein folding, structure, and stability (8, 9). In addition, homologs of RNase A have important cytotoxic and antitumor properties (10). The rate-limiting step for the RNase A reaction is a protein conformational change that is coupled to the product release step (1, 11). This conformational change involves multiple amino acid residues throughout the protein, including those distant from the active site (12-14). These mobile regions include two loops: loop 1,...

Insights into protein flexibility: The relationship between normal modes and conformational change upon protein–protein docking

“…This mechanism is supported by other studies where the unbound protein was found to sample conformations close to the bound form (5)(6)(7)34). However, our analysis shows that for only approximately one-third of the proteins with a large conformational change upon association, do the direction and location of motion along one of the lowest normal modes agree well with the observed conformational change.…”

Global structural motions from the strain of a single hydrogen bond

“…The suggested role of this azurin flexibility, or lack of rigid protein matrix, is to facilitate electron transfer (30). From this perspective, and from the growing evidence that active-site dynamics are critical for enzyme catalysis (31,32), it is notable that the H43F mutation has only a modest impact on SOD1 activity. One explanation might be that the activity of SOD1 is near diffusion control (7) and not limited by dynamics at the observed timescales.…”

How Do Enzymes Really Work?

“…Histidine 48 is at the top of the hinge, and its protonation state is important for this process. More direct evidence for the role of this residue in the conformational change of the isolated enzyme and in the catalytic reaction has been obtained recently with nuclear magnetic resonance methods (13). When the substrate binds, the hinge closes, squeezing out water and creating a hydrophobic environment.…”