Consequences of Heterogeneous Crowding on an Enzymatic Reaction: A Residence Time Monte Carlo Approach.

Translational diffusion of a free substrate in crowded metabolically active spaces such as cell cytoplasm or mitochondrial matrix is punctuated by collisions and nonspecific interactions with soluble/immobile macromolecules/macrostructures in a variety of shapes/sizes. It is not understood how such disruptions alter enzyme reaction kinetics in such spaces. A novel Monte Carlo (MC) technique, "residence time MC", has been developed to study the kinetics of a simple enzyme-substrate reaction in a crowded milieu using a single immobile enzyme in the midst of diffusing substrates and products. The reaction time lost while the substrate nonspecifically interacts or is transiently trapped with ambient macromolecules is quantified by introducing the residence time "tau". Tau scales with the size of crowding macromolecules but makes the knowledge of their shape redundant. The residence time thus presents a convenient parameter to realistically mimic the sticky surroundings encountered by a diffusing substrate in heterogeneously crowded physiological spaces. Results reveal that for identical substrate concentration and excluded volume, increase in tau significantly diminished enzymatic product yield and reaction rate, slowed down substrate/product diffusion, and prolonged their relaxation times. A smooth transition from the anomalous subdiffusive motion to normal diffusion at long time limits was observed irrespective of the value of tau. The predictions from the model are shown to be in qualitative agreement with in vitro experimental data revealing the rate of alkaline phosphatase-catalyzed hydrolysis of