Studies on Single Alkaline Phosphatase Molecules:  Reaction Rate and Activation Energy of a Reaction Catalyzed by a Single Molecule and the Effect of Thermal DenaturationThe Death of an Enzyme

Abstract

Single molecules of alkaline phosphatase are captured in a capillary filled with a fluorogenic substrate. During incubation, each enzyme molecule creates a pool of fluorescent product. After incubation, the product is swept through a high-sensitivity laser-induced fluorescence detector; the area of the peak provides a precise measure of the activity of each molecule. Three studies are performed on captured enzyme molecules. In the first study, replicate incubations are performed on the same molecule at constant temperature; the amount of product increases linearly with incubation time. Single enzyme molecules show a range of activity; the most active molecules have over a 10-fold higher activity than the least active molecules. In the second study, replicate incubations are performed on the same molecule at successively higher temperatures. The activation energy of the reaction catalyzed by a single molecule is determined with high precision. Single enzyme molecules show a range of activation energy; microheterogeneity extends to thermodynamic properties of catalysis. The average activation energy is within experimental error of the activation energy obtained from analysis of a bulk sample. These results are consistent with the first postulate of statistical thermodynamics: a thermodynamic property obtained from the time average of an individual molecule is identical to that produced by an ensemble average over a large number of molecules. In the third study, the activity of single enzyme molecules is measured after partial heat denaturation. The number of active molecules decreases in proportion to the extent of denaturation. However, the activity of the surviving molecules is experimentally indistinguishable from the activity of control enzyme. Thermal denaturation of alkaline phosphatase is a catastrophic process, wherein the molecule undergoes irreversible conversion to an inactive form.

ATP turnover by individual myosin molecules hints at two conformers of the myosin active site

“…S2). Thus, in the present study, we could not detect intermolecular (static) variation as in other single molecule enzyme studies (14)(15)(16). Instead, the variability observed here is due to the limited number of events we could record from individual molecules.…”

“…variation seen for other enzymes (14)(15)(16). Kinetic modeling suggested that the two dwell time populations originate from two active site conformers with different ATP binding features.…”

“…Here we explored the feasibility to study ATPase kinetics from individual myosin molecules by total internal reflection fluorescence (TIRF) microscopy using fluorescently labeled ATP as used in previous studies to follow ATP turnover by motor proteins (10)(11)(12)(13). One aim was to see whether myosin molecules show a similar variability in kinetic parameters among individual molecules as previously found in single molecule studies for other enzymes (14)(15)(16). Such variability among individual molecules would limit, even in single molecule studies, detection of subtle but relevant changes in ATPase kinetics by, for example, posttranslational modifications.…”

Single‐Molecule Fluorescence Studies of Protein Folding and Conformational Dynamics

“…Further analysis using higher order correlation functions did not help discrimination between this simple model involving two alternative conformations and another involving a continuum of conformers. 195 This seminal study definitely confirmed the existence of static and dynamic heterogeneity in enzymes, 196,197 illustrating the power of single-molecule fluorescence studies for elucidating enzymatic reaction mechanisms inaccessible via ensemble methods.…”

Optical Studies of Single Molecules at Room Temperature

“…The heterogeneity was found to be static on the hour timescale because the same enzyme molecule produces the same zone intensity after another incubation period. Using a similar approach, Craig et al studied single alkaline phosphatase molecules and found an even broader, multipeak distribution of activities (83). The heterogeneity was attributed to glycosylation and other posttranslational modifications, which produce nonidentical copies of enzyme in this system.…”