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A microfluidic device capable of storing picoliter droplets containing single bacteria at constant volumes has been fabricated in PDMS. Once captured in droplets that remain static in the device, bacteria express both a red fluorescent protein (mRFP1) and the enzyme, alkaline phosphatase (AP), from a biscistronic construct. By measuring the fluorescence intensity of both the mRFP1 inside the cells and a fluorescent product formed as a result of the enzymatic activity outside the cells, gene expression and enzymatic activity can be simultaneously and continuously monitored. By collecting data from many individual cells, the distribution of activities in a cell is quantified and the difference in activity between two AP mutants is measured.

Phosphoryl transfer reactions are ubiquitous in biology and metal fluoride complexes have played a central role in structural approaches to understanding how they are catalyzed. In particular, numerous structures of AlFx-containing complexes have been reported to be transition state analogs (TSAs). A survey of nucleotide kinases has proposed a correlation between the pH of the crystallization solution and the number of coordinated fluorides in the resulting aluminum fluoride TSA complexes formed. Enzyme ligands crystallized above pH 7.0 were attributed to AlF3, whereas those crystallized at or below pH 7.0 were assigned as AlF4-. We use 19F NMR to show that for beta-phosphoglucomutase from Lactococcus lactis, the pH-switch in fluoride coordination does not derive from an AlF4- moiety converting into AlF3. Instead, AlF4- is progressively replaced by MgF3- as the pH increases. Hence, the enzyme prioritizes anionic charge at the expense of preferred native trigonal geometry over a very broad range of pH. We demonstrate similar behavior for two phosphate transfer enzymes that represent typical biological phosphate transfer catalysts: an amino acid phosphatase, phosphoserine phosphatase from Methanococcus jannaschii and a nucleotide kinase, phosphoglycerate kinase from Geobacillus stearothermophilus. Finally, we establish that at near-physiological ratios of aluminum to magnesium, aluminum can dominate over magnesium in the enzyme-metal fluoride inhibitory TSA complexes, and hence is the more likely origin of some of the physiological effects of fluoride.

The efficient integration of binding, catalysis, and multiple turnovers remains a challenge in building enzyme models. We report that systematic derivatization of polyethylene imine (PEI) with alkyl (C(2)-C(12)), benzyl, and guanidinium groups gives rise to catalysts ('synzymes') with rate accelerations (k(cat)/k(uncat)) of up to 10(4) for the intramolecular transesterification of 2-hydroxypropyl-p-nitrophenyl phosphate, HPNP, in the absence of metal. The synzymes exhibit saturation kinetics (K(M) approximately 250 microM, k(cat) approximately 0.5 min(-1)) and up to 2340 turnovers per polymer molecule. Catalysis can be specifically and competitively inhibited by anionic and hydrophobic small molecules. The efficacy of catalysis is determined by the PEI derivatization pattern. The derivatization reagents exert a synergistic effect, i.e., their combinations increase catalysis by more than the sum of each single modification. The pH-rate profile for k(cat)/K(M) is bell shaped with a maximum at pH 7.85 and can be explained as a combination of two effects that both have to be operative for optimal activity: K(M) increases at high pH due to deprotonation of PEI amines that bind the anionic substrate and kcat decreases as the availability of hydroxide decreases at low pH. Thus, catalysis is based on substrate binding by positively charged amine groups and the presence of hydroxide ion in active sites in an environment that is tuned for efficient catalysis. Inhibition studies suggest that the basis of catalysis and multiple turnovers is differential molecular recognition of the doubly negatively charged transition state (over singly charged ground state and product): this contributes a factor of at least 5-10-fold to catalysis and product release.

Two independent investigations of catalysis by BSA and other serum albumins are combined to provide detailed insight into the mechanism of a classical proton-transfer reaction taking place on the surface of a protein. The Kemp elimination involves the general base-catalyzed removal of a proton from carbon and is known to be highly sensitive to medium effects. The serum albumins bind and catalyze the eliminative fragmentation of 5-nitrobenzisoxazole with remarkable efficiency and “accidental specificity.” A binding site and a likely catalytic lysine are identified, and factors contributing to the efficiency of catalysis analyzed. A key factor appears to be a differentiated microenvironment, which allows delocalized negative charge to develop in a stabilizing hydrophobic pocket next to a polar region where the developing ammonium cation is not disfavored. Catalytic efficiency is discussed in terms of effective molarities for the reaction catalyzed by serum albumins and by catalytic antibodies and compared w...

We report a second catalytic activity of Pseudomonas aeruginosa arylsulfatase (PAS). Besides hydrolyzing sulfate monoesters, this enzyme catalyzes the hydrolysis of phosphate monoesters with multiple turnovers (90), a k(cat) value of 0.023 s(-1), a K(M) value of 29 microM, and a kcat/K(M) ratio of 790 M(-1) s(-1) at pH 8.0. This corresponds to a remarkably high rate acceleration of 10(13) relative to the nonenzymatic hydrolysis [(k(cat)/K(M))/k(w)] and a transition-state binding constant (K(tx)) of 3.4 pM. Promiscuous phosphatase and original sulfatase activities only differ by a factor of 620 (measured by k(cat)), so the enzyme provides high accelerations for both reactions. The magnitudes and relative similarity of the kinetic parameters suggest that a functional switch from sulfatase to phosphatase activities is feasible, either by gene duplication or by direct evolution via an intermediate enzyme with dual specificity.

Enzyme catalysis depends on subtle combinations of effects that are difficult to separate and quantify.1 Medium effects are a crucial part of this package, but the assignment of a local dielectric constant to the structured microenvironment of an active site and its effect on ground state destabilization2 or transition state stabilization by electrostatic interactions3 is experimentally impossible. We aim to mimic and thus begin to quantify such active site medium effects experimentally. As a test reaction, we use the eliminative cleavage of benzisoxazole (1 f 2, the Kemp elimination), known to be particularly sensitive to the effects of the medium.4,5 We report that a subset