Single enzyme pathways and substrate fluctuations

The ability to dynamically probe single enzymes allows the experimental investigation of enzyme kinetics with unprecedented resolution. In this paper we develop a simple theory which predicts that certain classes of enzyme pathways can be distinguished by studying the turnover rate, V , as a function of substrate concentration, [S]. In particular, we study the steady state of a single enzyme interacting with a bath of substrate molecules, and analyse it as a stochastic process. The V ([S]) relation is found to depend sensitively on the manner in which substrate molecules in the bath are replenished. We focus on a gedanken experiment in which the average substrate concentration is kept fixed by allowing molecules to enter the bath at a constant rate. We derive the exact relationship between V and [S], which has a relatively simple form, though different to that of the Michaelis–Menten (MM) equation. Interestingly, the MM equation is exactly recovered if the substrate concentration is instantaneously maintained with molecular precision. We examine the new V ([S]) relation for a number of enzyme pathways and find that it differentiates between enzyme reactions involving one or many intermediate enzyme–substrate complexes. This, in principle, allows one to probe the internal conformations of enzymes by careful measurement of V ([S]) curves in appropriately designed single enzyme experiments. PACS numbers: 82.39.−k, 87.15.Rn, 05.40.−a