Catalytic and binding poly-reactivities shared by two unrelated proteins: The potential role of promiscuity in enzyme evolution

Abstract

It is generally accepted that enzymes evolved via gene duplication of existing proteins. But duplicated genes can serve as a starting point for the evolution of a new function only if the protein they encode happens to exhibit some activity towards this new function. Although the importance of such catalytic promiscuity in enzyme evolution has been proposed, little is actually known regarding how common promiscuous catalytic activities are in proteins or their origins, magnitudes, and potential contribution to the survival of an organism. Here we describe a pattern of promiscuous activities in two completely unrelated proteins-serum albumins and a catalytic antibody (aldolase antibody 38C2). Despite considerable structural dissimilarities-in the shape of the cavities and the position of catalytic lysine residues-both active sites are able to catalyze the Kemp elimination, a model reaction for proton transfer from carbon. We also show that these different active sites can bind promiscuously an array of hydrophobic negatively charged ligands. We suggest that the basic active-site features of an apolar pocket and a lysine residue can act as a primitive active site allowing these promiscuous activities to take place. We also describe, by modelling product formation at different substrate concentrations, how promiscuous activities of this kind- inefficient and rudimentary as they are-can provide a considerable selective advantage and a starting point for the evolution of new functions.

Fig. 1.

Catalysis of the elimination benzisoxazole 1 by antibody 38C2. Substrate (1, 40 μM) was added to 50 mM Tris buffer (pH 8.9) or to the same buffer containing antibody 38C2 (4.4 μM). The rate of elimination was followed by monitoring the appearance of the phenol product 2 at 405 nm.

Fig. 2.

The kinetic parameters for the 38C2-catalyzed elimination of benzisoxazole 1. Initial rates of product release (v_o) were determined at a range of substrate concentrations ([S_o] = 20–500 μM; [Ab_o] = 4.4 μM; in 50 mM Tris at pH 8.9; T = 25°C) Data were fitted directly to the Michaelis-Menten model (v_o = [Ab_o] × k_cat × [S_o]/([S_o] + K_M) and also to the Eadie-Hofstee fit (inset) to give k_cat of 1.56 ± 0.02/min and K_M of 70.9 ± 3.3 μM.

Fig. 3.

The kinetic pK_a for the 38C2-catalyzed elimination of benzisoxazole 1. Initial rates of product release (v_o) were determined at pH 8.2–10.4 under pseudo-first-order conditions ([S_o] = 25 μM, [Ab₀] = 4.4 μM, Buffer: 50 mM Tris at pH 7.4–8.9, 50 mM AMP at pH 9.15–10.4, T = 25°C). Data were fit to the equation: , where 1/(10^(pKa−pH) + 1) is the active fraction of antibody at a given pH, v_o^H is the rate of the fully active (deprotonated) form of 38C2 (4.17 ± 0.1 μM/min) and pK_a is 8.9 ± 0.04.

Fig. 4.

Secondary structures and surface representations of the active sites of aldolase antibody 33F12 (a homolog of 33C2) (Barbas et al. 1997) and HSA (Curry et al. 1998). (a) The active-site surface of 33F12 takes the form of a cleft with the catalytic lysine residue (H93) at the bottom. (b) The main chain and residues surrounding Lys 93 (in yellow) and forming the cavity are depicted (in green) (H33W, L96Y, L27dH, L98F, L32F, H103W, H98Y, H93K, H95Y); these come from the β-strands of both the heavy (H) and the light chain (L). (c) The IIA site of HSA consists of a deep channel into which the ɛ-amino group of Lys 199 projects. (d) The main chain of the α-helices and the side chains forming the walls of the IIA site are depicted (in green and lysine in yellow) (V241, L238, L219, F223, L234, I264, I290, A261, L260, K199). PDB files 1AXT (Barbas et al. 1997) and 1BKE (Curry et al. 1998) were used for depicting the sites of aldolase antibody 33F12 and HSA, respectively. Surfaces (A and C) were calculated using a 1.4 Å probe in GRASP (Nicholl and Honig 1991). Electrostatic potentials are marked, with red corresponding to an overall negative charge and blue for positive. Ribbon representations were created using SETOR (Evans 1993).

Fig. 5.

The putative enrichment for product in the presence of promiscuous catalysts HSA and antibody 38C2. The enrichment is the ratio of product obtained in the presence versus the absence of a promiscuous catalyst, at different substrate concentrations. Assuming initial velocity conditions ([Product] ≤ 0.1[S_o]), the enrichment is given by: [E_o](k_cat/k_buffer)/([S_o] + K_M); where [E_o] is the concentration of the promiscuous catalyst (10 μM; see text); k_cat and K_M are the kinetic parameters for HSA and 38C2 (Table 1); and, k_buffer is the first-order rate constant for the background, buffer-catalyzed reaction under the same conditions. Results are presented for two ranges of substrate concentrations: [S_o] = 1–150 μM (A) and [S_o] = 2–10 mM (B).