Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O-Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism

Abstract

By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4-6 log-fold (10-10) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT-OASS (cysteine regulatory complex) follows a noncanonical "facilitated dissociation" mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that "competitive allostery" is the general feature of noncanonical "facilitated/accelerated dissociation" mechanisms. Further understanding of the mechanistic framework of "competitive allosteric" mechanism may allow us to design a new family of "competitive allosteric drugs/small molecules" that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

Molecular mechanism of selective substrate engagement and inhibitor disengagement of cysteine synthase

“…Further, both interact to form a stable multi-enzyme complex, referred to as Cysteine Regulatory Complex (CRC) [15][16][17]. Our recent studies showed that CRC complex dissociates in the presence of OAS, the substrate of CS, at stoichiometric concentrations [1,18]. Dissociation of CRC at stoichiometric concentration of OAS was not expected as previous studies reported that affinity of SAT (inhibitor) is 4-6 log-fold higher than that of OAS [14][15][16][17][18].…”

“…Proteases accomplish substrate-specificity through specific sub-pockets (S1, S2), mostly non-catalytic residues, distributed within the active site [10,13]. Similarly, the active site of cysteine synthesis enzyme (CS) is mapped into three sub-pockets (S1, S2, S3) that coordinate the substrateinduced conformational transition to closed state [1]. In a recent study, we showed that substrate-binding induced active site conformational changes allowed CS to selectively recruit its substrate, O-acetyl serine (OAS), in the presence of high affinity natural inhibitor, the C-terminal of serine acetyl transferase (SAT).…”

“…Our extensive structural and analytical work revealed that only substrate-binding can induce the active site of CS to a closed-state, not the binding of the high affinity inhibitor. We proposed a novel "competitive-allosteric" mechanism by which CS selectively recruits its substrate while bound to high-affinity natural inhibitors [1]. In this work, we traced the molecular origins of substrate selectivity by employing a combination of approaches and provide evidences for substrate selectivity.…”

Force-Dependent Facilitated Dissociation Can Generate Protein-DNA Catch Bonds

“…Recent work with a variety of biomolecules such as transcription factors (20,22,(24)(25)(26)(27)(28)(29), metalloregulators (21,30), chromatin effectors (31), DNA polymerases (32)(33)(34)(35)(36), antibody-antigen complexes (37), and other biological complexes (23,(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48) has demonstrated that facilitated dissociation, in which protein dissociation rates depend on ambient protein or other biomolecule concentration, is a widespread phenomenon both in vitro and in vivo. Facilitated dissociation contrasts with classical kinetic models of bimolecular complexes that undergo simple, binary on/ off transitions and have concentration-dependent association rates and concentration-independent dissociation rates.…”

Force-dependent facilitated dissociation can generate protein-DNA catch bonds

“…Recent work with a variety of biomolecules, such as transcription factors (20,22,(24)(25)(26)(27)(28)(29), metalloregulators (21,30), chromatin effectors (31), DNA polymerases (32)(33)(34)(35)(36), antibody-antigen complexes (37), and other biological complexes (23,(38)(39)(40)(41)(42)(43)(44)(45)(46)(47)(48) has demonstrated that facilitated dissociation, in which protein dissociation rates depend on ambient protein or other biomolecule concentration, is a widespread phenomenon both in vitro and in vivo. Facilitated dissociation contrasts with classical kinetic models of bimolecular complexes that undergo a simple, binary on/off transitions, and have concentration-dependent association rates and concentration-independent dissociation rates.…”