A Quantitative Index of Substrate Promiscuity

Abstract

Catalytic promiscuity is a widespread, but poorly understood, phenomenon among enzymes with particular relevance to the evolution of new functions, drug metabolism, and in vitro biocatalyst engineering. However, there is at present no way to quantitatively measure or compare this important parameter of enzyme function. Here we define a quantitative index of promiscuity (I) that can be calculated from the catalytic efficiencies of an enzyme toward a defined set of substrates. A weighted promiscuity index (J) that accounts for patterns of similarity and dissimilarity among the substrates in the set is also defined. Promiscuity indices were calculated for three different enzyme classes: eight serine and cysteine proteases, two glutathione S-transferase (GST) isoforms, and three cytochrome P450 (CYP) isoforms. The proteases ranged from completely specific (granzyme B, J ) 0.00) to highly promiscuous (cruzain, J ) 0.83). The four drug-metabolizing enzymes studied (GST A1-1 and the CYP isoforms) were highly promiscuous, with J values between 0.72 and 0.92; GST A4-4, involved in the clearance of lipid peroxidation products, is moderately promiscuous (J ) 0.37). Promiscuity indices also allowed for studies of correlation between substrate promiscuity and an enzyme's activity toward its most-favored substrate, for each of the three enzyme classes.

Enzymatic Detoxication, Conformational Selection, and the Role of Molten Globule Active Sites

“…The ␣-class GST family includes the isoforms GSTA1-1 and A4 -4, which are highly promiscuous and highly substrate specific, respectively, despite their high sequence similarity and nearly superimposable structures (Fig. 2, a and b) (12,18,19). Whereas GSTA1-1 is an archetypal promiscuous detoxication enzyme, GSTA4 -4 has great specificity for straight chain unsaturated lipid aldehydes, about nine carbons in length, that result from lipid peroxidation (20).…”

Ensemble Perspective for Catalytic Promiscuity

“…the biological importance of their promiscuity, and this represents a change in a longstanding biological paradigm, wherein "specificity" was considered to be the defining feature of proteins, enzymes, and metabolic pathways (1)(2)(3)(4)(5)(6). It is now clear that functionally useful promiscuity plays a role in the immune system (7)(8)(9)(10), in signal transduction pathways (11), and in enzymatic detoxification (12)(13)(14)(15).…”

“…The homodimeric GSTA1-1 is an archetypal detoxification enzyme with extreme catalytic promiscuity (5,12). As a predominantly hepatic enzyme, it plays a role in chemical detoxification by conjugating glutathione (GSH) to an extraordinary range of electrophilic substrates (12,(23)(24)(25)(26)(27).…”

“…GSTA4-4 has a static C-terminal helix localized along one edge of the active site that does not undergo any rearrangement upon binding substrate (31,32). The GSTA1-1/GSTA4-4 pair has been a powerful model for engineering substrate specificity and understanding its structural basis in GSTA4-4 (5,12,33,34), but the molecular basis for the contrasting promiscuity of GSTA1-1 is not understood in any thermodynamic context. Therefore, we have performed scanning calorimetry with these GST isoforms and analyzed the results with a modified Landau theory of phase transitions, as described for protein folding (35,36).…”

Cys-X Scanning for Expansion of Active-site Residues and Modulation of Catalytic Functions in a Glutathione Transferase

“…Altered Substrate Selectivity Profiles via Diverse Cys-X Substitutions-Glutathione transferases are generally promiscuous enzymes, as indicated by the promiscuity index (19), and they accept a wide range of alternative substrates representing many different types of reactions. For biotechnological applications, such as fine chemical synthesis, promiscuity will not be a desirable trait for an engineered enzyme.…”