How enzymes harness highly unfavorable proton transfer reactions

Abstract

Acid-base reactions that are exceedingly unfavorable under standard conditions can be catalytically important at enzyme active sites. For example, in triose phosphate isomerase, a glutamate side chain (nominal pK a ≈ 4 in solution) can in fact deprotonate a CH group that is vicinal to a carbonyl (pK a ≈ 18 in solution). This is true because of three distinct interactions: (a) ground state pK a shifts due to environment polarity and electrostatics; (b) dramatic increases in effective molarity due to optimization of proximity and orientation; and (c) transition state pK a shifts due to binding interactions and the formation of strong low barrier hydrogen bonds. In this report, we review the literature showing that the sum of these three effects supplies more than enough free energy to push forward proton transfer reactions that under standard conditions are exceedingly nonspontaneous and slow.

The Proton in Biochemistry: Impacts on Bioenergetics, Biophysical Chemistry, and Bioorganic Chemistry

“…Such a low-barrier H-bond has in fact been confirmed in the histidine-serine active site pair in chymotrypsin and other serine proteases (Frey et al, 1994). Formation of such super-strong H-bonds could supply 7-25 kcal/mol toward lowering the activation energy of the proton transfer step (Silverstein, 2021).…”

“…Thus, if we define for any acid-base reaction ΔpK a ≡ pK a (HB + ) -pK a (HA), then the reaction will be spontaneous if ΔpK a is positive. 5 With this in mind, it is quite surprising that acid-base reactions featured in enzyme catalysis can be exceedingly nonspontaneous, with some ΔpK a values ranging from −8 to −23 (Silverstein, 2021). The question of how such incredibly nonspontaneous reactions can be catalytically useful will be discussed below.…”

“…How could such incredibly nonspontaneous reactions be catalytically useful? As discussed recently (Silverstein, 2021), to get around this problem, enzymes employ several catalytic strategies that can be categorized as either ground state or transition state effects. The first ground state effect is the alteration of pK a by local environment that we discussed above.…”

“…The first ground state effect is the alteration of pK a by local environment that we discussed above. For example, carbon acid substrates bound to the enzyme can have pK a values 4-20 units lower than in aqueous solution; increases in base strength of 2-5 units are also found (Silverstein, 2021). Some of these nonspontaneous proton transfers can be brought to ΔG °≈ 0 by this pK a shift effect alone, but more generally, about 2/3 of the unfavorable free energy is supplied by this effect; this leaves about 4-14 kcal/ mol to be supplied by other effects (Silverstein, 2021).…”

“…Menger proposed a split-site model of enzyme catalysis that allows calculation of the amount of spontaneous substrate binding free energy that can be applied to lowering the activation energy (Menger, 1993) (Menger, 2005). Readers are referred to these original papers for the details of this useful model, but suffice it to say here that this effect has been estimated to lower the activation energy by 6-18 kcal/mol (Silverstein, 2021). Additionally, Cleland proposed that normal ground state hydrogen bonds can become shorter, stronger low-barrier hydrogen bonds in the transition state (Cleland and Kreevoy, 1994) (Cleland et al, 1998) (Cleland, 2000).…”