Enzyme I of the Phosphotransferase System: Induced-Fit Protonation of the Reaction Transition State by Cys-502†
journal contributionposted on 02.04.2003, 00:00 by Luis Fernando García-Alles, Ignacio Alfonso, Bernhard Erni
Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal domain with the phosphorylation site (His-189) and a C-terminal domain with the PEP binding site. Here we use C3-substituted PEP analogues as substrates and inhibitors and the EI(C502A) mutant to characterize structure−activity relationships of the PEP binding site. EI(C502A) is 10 000 times less active than wild-type EI [EI(wt)] with PEP as the substrate, whereas the two forms are equally active with ZClPEP. Cys-502 acts as an acid−base catalyst which stereospecifically protonates the pyruvoyl enolate at C3. The electron-withdrawing chlorine of ZClPEP can compensate for the lack of Cys-502, and in this case, the released 3-Cl-enolate is protonated nonstereospecifically. Several PEP analogues were assayed as inhibitors and as substrates. The respective KI/Km ratios vary between 3 and 40 for EI(wt), but they are constant and around unity for EI(C502A). EI(wt) with PEP as the substrate is inhibited by oxalate, whereas EI(C502A) with ZClPEP is not. The different behavior of EI(wt) and EI(C502A) toward the PEP analogues and oxalate suggests that the PEP binding site of EI(wt) exists in a “closed” and an “open” form. The open to closed transition is triggered by the interaction of the substrate with Cys-502. The closed conformation is sterically disfavored by C3-modified substrate analogues such as ZClPEP and ZMePEP. If site closure does not occur as with EI(C502A) and bulky substrates, the transition state is stabilized by electron dispersion to the electron-withdrawing substituent at C3.