Uniform binding and negative catalysis at the origin of enzymes

Abstract

Enzymes are well known for their catalytic abilities, some even reaching "catalytic perfection" in the sense that the reaction they catalyze has reached the physical bound of the diffusion rate. However, our growing understanding of enzyme superfamilies has revealed that only some share a catalytic chemistry while others share a substrate-handle binding motif, for example, for a particular phosphate group. This suggests that some families emerged through a "substrate-handle-binding-first" mechanism ("binding-first" for brevity) instead of "chemistry-first" and we are, therefore, left to wonder what the role of non-catalytic binders might have been during enzyme evolution. In the last of their eight seminal, back-to-back articles from 1976, John Albery and Jeremy Knowles addressed the question of enzyme evolution by arguing that the simplest mode of enzyme evolution is what they defined as "uniform binding" (parallel stabilization of all enzyme-bound states to the same degree). Indeed, we show that a uniform-binding proto-catalyst can accelerate a reaction, but only when catalysis is already present, that is, when the transition state is already stabilized to some degree. Thus, we sought an alternative explanation for the cases where substrate-handle-binding preceded any involvement of a catalyst. We find that evolutionary starting points that exhibit negative catalysis can redirect the reaction's course to a preferred product without need for rate acceleration or product release; that is, if they do not stabilize, or even destabilize, the transition state corresponding to an undesired product. Such a mechanism might explain the emergence of "binding-first" enzyme families like the aldolase superfamily.

Keywords: enzyme evolution; negative catalysis; primordial catalyst; substrate-handle; triose phosphate isomerase; uniform binding.

Wayne (left) and Danny (right) discussing uniform binding at Temple Stream, New Zealand (photo credit: Prof. Joel Mackay, University of Sydney)

FIGURE 1

The chemistry‐first versus substrate‐handle‐binding‐first modes of emergence and the evolutionary origin of triose phosphate isomerase. (a) Emergence by chemistry‐first is demonstrated by the enolase family, members of which share a key chemical step—abstraction of a proton next to a carboxylate (shown in green). (b) In contrast, triose phosphate isomerase belongs to the aldolase superfamily, in which nearly all family members act on substrates that contain a phosphate handle—that is, a phosphate group that serves in substrate binding but is not part of the reaction. (c,d) The two modes of emergence are also reflected in the way by which new family members diverge from existing ones. (c) Chemistry‐first is manifested in recruitment via acceptance of alternative, non‐native substrates that make use of pre‐existing catalytic machinery to perform a similar reaction on the new substrate (substrate ambiguity). (d) In contrast, in catalytic promiscuity, binding of a new substrate with the same handle results in the enzyme promiscuously catalyzing a reaction that differs from the native one.

FIGURE 2

Phosphate binding in the primordial protein world. Shown are various phosphate binding elements, which appear in protein superfamilies that emerged prior to the LUCA. These elements have key characteristics that coincide with early emergence. First, phosphate binding is realized within a short segment comprising 3–5 residues that provides a “nest” of hydrogen bonds. The primordial binding segments reside mostly at the N‐termini of an ɑ‐helix, as observed in the HhH, Rossmann, and P‐loop NTPase lineages, as well as in the aldolase superfamily to which TPI belongs. Second, phosphate binding is preferentially mediated by abiotic amino acids, foremost by glycine and serine/threonine (the latter often form bidentate hydrogen bonds with the backbone NH and side‐chain OH groups). Note that the phosphate‐binding site of the aldolase superfamily is traditionally assigned to the β‐strands (β7, β8) of the TIM barrel; however, the N‐terminus of a short helix is a critical part.

FIGURE 3

Uniform binding does not accelerate the rate of an uncatalyzed reaction. (a) Substrate S is spontaneously converted to P via a single transition state S^‡, quantified by the equilibrium constant K ₁. The rate of product formation from S^‡ is given by k ^‡ which is directly proportional to the frequency of the vibrational mode responsible for converting the activated complex to P. A uniform binder U (e.g., a peptide) binds to S, S^‡, and P with equilibrium constants K _S  = K _S‡ = K _P (since uniform binding entails equal equilibrium binding constants). (b) Free energy profile for the system shown in. Since all three states are equally stabilized by binding to U, the activation energies of both the bound and unbound reactions are equal (K ₁ = K ₁ ^UB ). (c–e) Simulation of the model assuming the binding constants are (c) K _S  = K _S‡ = K _P  = 10⁻⁴ μM⁻¹, (d) 10⁻² μM⁻¹, or (e) 1 μM⁻¹. In all three cases, the total amount of product (P _tot  = P + UP, dot‐dashed black line) is the same, regardless of the strength of binding by U.

FIGURE 4

Diversion of a reaction's outcome by uniform binding and negative catalysis. (a) The substrate, S, can react via two different trajectories to give two alternative products, either P₁ or P₂. As depicted in a free energy diagram, the dominance of P₂ in solution is due to a lower transition energy which translates to faster kinetics (K ₂ > K ₁). (b) The same reaction, mediated via a uniform binder. The free energies of the corresponding non‐bound species, S, S^‡ _1/2, and P_1/2, are shown as dashed gray lines, and the outcome of uniform stabilization (an equal reduction in their free energies) as gray arrows. Uniform binding dictates that the transition energy (from U·S to U·S^‡ _1/2) remains the same, and therefore that K ₂ ^UB  = K ₂ and K ₁ ^UB  = K ₁. Consequently, K ₂ ^UB  > K ₁ ^UB and P₂ remains the dominant product. (c) Q is similar to U with regard to the energy profile of the S → P₁ reaction, but performs negative catalysis with regards to the S → P₂ reaction. This is depicted as a relative destabilization of Q·S^‡ ₂ and Q·P₂ compared to U·S^‡ ₂ and U·P₂ (as shown by the light‐green arrows). Consequently, the barrier to P₂ is higher than for P₁, making P₁ the dominant product (K ₁ ^UB  > K ₂ ^NC ). (d) Diagram of a setting where negative catalysis is desirable. In solution, the dominant reaction S → P₂ might require water or metal ions. The desirable reaction S → P₁ is much slower and so P₁ is rarely produced. (e) Partial exclusion of solvent is a possible mechanism for negative catalysis. Binding sequesters S, but has the side effect of excluding solvent components that promote the S → P₂ reaction, that is, slowing the undesirable reaction by raising the Q·S^‡ ₂ barrier (negative catalysis, panel c). If S → P₁ is unaffected, P₁ becomes the dominant product.

FIGURE 5

Negative catalysis of a reaction leading to an undesirable product. (a) The model assumes that a substrate can react to give either the product P₁, which is the desired but disfavored product, or P₂ which is favored in solution (k ₂ ^non  = 10 k ₁ ^non ). (b) In the presence of a uniform binder that does not alter the rate of formation of P₁ nor of P₂, (corresponding to Figure 4b, where Q is actually U in this case), the P₁:P₂ ratio remains 1:10, as in solution. Note, however, that the outcomes are the corresponding product complexes Q·P_1/2 while the concentrations of the unbound products P_1/2 are effectively nil. (c) The product ratio changes if the binder stabilizes the transition state leading to P₂ to a lesser extent than the transition state leading to P₁, here by 1.4 kcal mol⁻¹. This results in a 10‐fold reduction in the rate of S‐to‐P₂ conversion when the substrate is bound to the binder (k ₂ ^NC  = 10⁻³ s⁻¹ compared to k ₂ ^non  = 10⁻² s⁻¹). The outcome under these conditions is P₁:P₂ ≈ 1. (d) This time, corresponding to Figure 4c, a 2.8 kcal mol⁻¹ differential destabilization of S₂ ^‡ leads to a 100‐fold rate reduction for S‐to‐P₂. The result is P₁ now predominating over P₂, at a ratio of P₁:P₂ ≈ 10:1.

FIGURE 6

The effect of coupled reactions and kinetic trapping of the product. (a) The reaction scheme used to simulate coupled reactions. The scheme is the same as in Figure 5, with the uniform binding equilibrium constants K _S  = K ₁ = K ₂ = 10⁴ μM⁻¹, and a 1,000‐fold slower rate of conversion to P₂ for the bound substrate compared to unbound, namely k ₂ ^NC  = 10⁻⁵ s⁻¹ versus k ₂ ^non  = 10⁻² s⁻¹. As before, for P₁ there is no difference between the rates: k ₁ ^non  = k ₁ ^UB  = 10⁻³/s. The binder's product, P₁, further reacts irreversibly to give the final product P*, with a forward rate constant, k _f , of 0.1 s⁻¹. (b) The concentrations of the different species as a function of time during the first 100 min, during which almost all of the substrate is converted to P₁ and is bound to Q. (c) The concentrations of the different species in longer time‐scales, where P* becomes the dominant species and Q returns to its unbound state.