Deciphering Binding Site Conformational Variability of Substrate Promiscuous and Specialist Enzymes.

Substrate promiscuity is the ability of enzymes to catalyze the same reaction with alternate substrates beyond their physiologically relevant cognate substrates. These promiscuous activities expand enzymes' functional landscape, enabling evolution to catalyze novel biochemical reactions or the rational design of enzymes. The molecular basis of substrate promiscuity remains elusive, although recent studies have highlighted the significance of active site conformational variability in imparting promiscuity through alternate accessible conformational states. Accordingly, it has been hypothesized that promiscuous enzymes can accommodate alternate substrates through their pre-existing conformations. Specialist enzymes, known to catalyze reactions with a particular substrate, may exhibit a predominant conformation for their native substrates. To explore the role of active site conformational flexibility in substrate promiscuity, we compare the conformational binding competent substates in substrate promiscuous and specialist enzymes by analyzing long-time explicit water molecular dynamics. In addition to visually analyzing the variability of alternate conformations, we quantitatively assess this using the native functionality score, which measures the similarity of each conformation to the native substrate-binding residues. Our analysis of the two enzymes each from promiscuous and specialist groups shows multiple and restrictive substrate-binding competent conformers in both groups. Thus, conformational dynamics likely play a key role in enabling both enzyme groups to bind different substrates, albeit to varying extents.