Structure-Based Predictive Models for Allosteric Hot Spots

In allostery, a binding event at one site in a protein modulates the behavior of a distant site. Identifying residues that relay the signal between sites remains a challenge. We have developed predictive models using support-vector machines, a widely used machine-learning method. The training data set consisted of residues classified as either hotspots or non-hotspots based on experimental characterization of point mutations from a diverse set of allosteric proteins. Each residue had an associated set of calculated features. Two sets of features were used, one consisting of dynamical, structural, network, and informatic measures, and another of structural measures defined by Daily and Gray [1]. The resulting models performed well on an independent data set consisting of hotspots and non-hotspots from five allosteric proteins. For the independent data set, our top 10 models using Feature Set 1 recalled 68–81% of known hotspots, and among total hotspot predictions, 58–67% were actual hotspots. Hence, these models have precision P = 58–67% and recall R = 68–81%. The corresponding models for Feature Set 2 had P = 55–59% and R = 81–92%. We combined the features from each set that produced models with optimal predictive performance. The top 10 models using this hybrid feature set had R = 73–81% and P = 64–71%, the best overall performance of any of the sets of models. Our methods identified hotspots in structural regions of known allosteric significance. Moreover, our predicted hotspots form a network of contiguous residues in the interior of the structures, in agreement with previous work. In conclusion, we have developed models that discriminate between known allosteric hotspots and non-hotspots with high accuracy and sensitivity. Moreover, the pattern of predicted hotspots corresponds to known functional motifs implicated in allostery, and is consistent with previous work describing sparse networks of allosterically important residues

Analysis of the cooperativity of human beta-cell glucokinase through the stimulatory effect of glucose on fructose phosphorylation.

Using overexpressed Escherichia coli sorbitol-6-phosphate dehydrogenase to monitor fructose 6-phosphate formation, we found that the stimulation of fructose phosphorylation by glucose was reduced in two human beta-cell glucokinase mutants with a low Hill coefficient or when the activity of wild type glucokinase was decreased by replacing ATP with poorer nucleotide substrates. Mutation of two other residues, neighboring glucose-binding residues in the catalytic site, also reduced the affinity for glucose as a stimulator of fructose phosphorylation. Among a series of glucose analogs, only 3, all substrates of glucokinase, stimulated fructose phosphorylation; other analogs were either inactive or inhibited glucokinase. Glucose increased the apparent affinity for inhibitors that are glucose analogs but not for the glucokinase regulatory protein or palmitoyl-CoA. These data indicate that the stimulatory effect of glucose on fructose phosphorylation reflects the positive cooperativity for glucose and is mediated by binding of glucose to the catalytic site. They support models involving the existence of two slowly interconverting conformations of glucokinase that differ through their affinity for glucose and for glucose analogs. We show by computer simulation that such a model can account for the kinetic properties of glucokinase, including the differential ability of mannoheptulose and N-acetylglucosamine to suppress cooperativity (Agius, L., and Stubbs, M. (2000) Biochem. J. 346, 413-421)

Purification and characterization of a microbial dehydrogenase - A vanillin : NAD(P)(+) oxidoreductase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M, value estimated by gel-filtration chromatography gave a value of 210,000. The enzyme made use of NAD(+) less effectively than NADP(+). Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD(+) or NADP(+). The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl2. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD(+) and NADP(+) protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD(+) or NADP(+). We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases

Study of the regulatory properties of glucokinase by site-directed mutagenesis: conversion of glucokinase to an enzyme with high affinity for glucose.

To identify the amino acids involved in the specific regulatory properties of glucokinase, and particularly its low affinity for glucose, mutants of the human islet enzyme have been prepared, in which glucokinase-specific residues have been replaced. Two mutations increased the affinity for glucose by twofold (K296M) and sixfold (Y214A), the latter also decreasing the Hill coefficient from 1.75 to 1.2 with minimal change in the affinity for ATP. Combining these two mutations with N166R resulted in a 50-fold decrease in the half-saturating substrate concentration (S0.5) value, which became then comparable to the Km of hexokinase II. The location of N166, Y214, and K296 in the three-dimensional structure of glucokinase suggests that these mutations act by favoring closure of the catalytic cleft. As a rule, mutations changed the affinity for glucose and for the competitive inhibitor mannoheptulose (MH) in parallel, whereas they barely affected the affinity for N-acetylglucosamine (NAG). These and other results suggest that NAG and MH bind to the same site but to different conformations of glucokinase. A small reduction in the affinity for the regulatory protein was observed with mutations of residues on the smaller domain and in the hinge region, confirming the bipartite nature of the binding site for the regulatory protein. The K296M mutant was found to have a threefold decreased affinity for palmitoyl CoA; this effect was additive to that previously observed for the E279Q mutant, indicating that the binding site for long-chain acyl CoAs is located on the upper face of the larger domain