A Method to Study Complex Enzyme Kinetics Involving Numerical Analysis of Enzymatic Schemes. The Mannitol Permease of Escherichia coli as an Example

An analysis of complex kinetic mechanisms is proposed that consists of two steps, (i) building of an kinetic scheme from experimental data other than steady-state kinetics and (ii) numerical simulation and analysis of the kinetics of the proposed scheme in relation to the experimental kinetics. Procedures are introduced to deal with large numbers of enzymatic states and rate constants, and numerical tools are defined to support the analysis of the scheme. The approach is explored by taking the mannitol permease of Escherichia coli as an example. This enzyme catalyzes both the transport of mannitol across the cytoplasmic membrane and the phosphorylation of mannitol. The challenge is to deduce the transport properties of this dimeric enzyme from the phosphorylation kinetics. It is concluded that (i) the steady-state kinetic behavior is largely consistent with the proposed catalytic cycle of the monomeric subunit, (ii) the kinetics provide no direct support but also do not disprove a coupled translocation of the binding sites on the two monomeric subunits. The approach reveals the need for further experimentation where the implementation of experimental results in the scheme conflict with the experimental kinetics and where specific experimental characteristics do not show up in the simulations of the proposed kinetic scheme.

Conserved Residues R420 and Q428 in a Cytoplasmic Loop of the Citrate/Malate Transporter CimH of Bacillus subtilis Are Accessible from the External Face of the Membrane

CimH of Bacillus subtilis is a secondary transporter for citrate and malate that belongs to the 2-hydroxycarboxylate transporter (2HCT) family. Conserved residues R143, R420, and Q428, located in putative cytoplasmic loops and R432, located at the cytoplasmic end of the C-terminal transmembrane segment XI were mutated to Cys to identify residues involved in binding of the substrates. R143C, R420C, and Q428C revealed kinetics similar to those of the wild-type transporter, while the activity of R432C was reduced by at least 2 orders of magnitude. Conservative replacement of R432 with Lys reduced the activity by 1 order of magnitude, by lowering the affinity for the substrate 10-fold. It is concluded that the arginine residue at position 432 in CimH interacts with one of the carboxylate groups of the substrates. Labeling of the R420C and Q428C mutants with thiol reagents inhibited citrate transport activity. Surprisingly, the cysteine residues in the cytoplasmic loops in both R420C and Q428C were accessible to the small, membrane-impermeable, negatively charged MTSES reagent from the external site of the membrane in a substrate protectable manner. The membrane impermeable reagents MTSET, which is positively charged, and AMdiS, which is negatively charged like MTSES but more bulky, did not inhibit R420C and Q428C. It is suggested that the access pathway is optimized for small, negatively charged substrates. Either the cytoplasmic loop containing residues R420 and Q428 is partly protruding to the outside, possibly in a reentrant loop like structure, or alternatively, a water-filled substrate translocation pathway extents to the cytoplasm-membrane interface.

Stereoselectivity of the membrane potential-generating citrate and malate transporters of lactic acid bacteria

The citrate transporter of Leuconostoc mesenteroides (CitP) and the malate transporter of Lactococcus lactis (MleP) are homologous proteins that catalyze citrate-lactate and malate-lactate exchange, respectively. Both transporters transport a range of substrates that contain the 2-hydroxycarboxylate motif, HO-CR2-COO- [Bandell, M., et al. (1997) J. Biol. Chem. 272, 18140-18146]. In this study, we have analyzed binding and translocation properties of CitP and MleP for a wide variety of substrates and substrate analogues. Modification of the OH or the COO- groups of the 2-hydroxycarboxylate motif drastically reduced the affinity of the transporters for the substrates, indicating their relevance in substrate recognition. Both CitP and MleP were strictly stereoselective when the R group contained a second carboxylate group; the S-enantiomers were efficiently bound and translocated, while the transporters had no affinity for the R-enantiomers. The affinity of the S-enantiomers, and of citrate, was at least 1 order of magnitude higher than for lactate and other substrates with uncharged R groups, indicating a specific interaction between the second carboxylate group and the protein that is responsible for high-affinity binding. MleP was not stereoselective in binding when the R groups are hydrophobic and as large as a benzyl group. However, only the S-enantiomers were translocated by MleP. CitP had a strong preference for binding and translocating the R-enantiomers of substrates with large hydrophobic R groups. These differences between CitP and MleP explain why citrate is a substrate of CitP and not of MleP. The results are discussed in the context of a model for the interaction between sites on the protein and functional groups on the substrates in the binding pockets of the two proteins.</p

The Conserved C-Terminus of the Citrate (CitP) and Malate (MleP) Transporters of Lactic Acid Bacteria Is Involved in Substrate Recognition

The membrane potential-generating transporters CitP of Leuconostoc mesenteroides and MleP of Lactococcus lactis are homologous proteins with 48% identical residues that catalyze citrate-lactate and malate-lactate exchange, respectively. The two transporters are highly specific for substrates containing a 2-hydroxycarboxylate motif (HO-CR2-COO-) in which substitutions of the R groups are tolerated well. Differences in substrate specificity between MleP and CitP are based on subtle changes in the interaction of the protein with the R groups affecting both binding and translocation properties. The conserved, 46-residue long C-terminal region of the transporters containing the C-terminal putative transmembrane segment XI was investigated for its role in substrate recognition by constructing chimeric transporters. Replacement of the C-terminal region of MleP with that of CitP and vice versa did not alter the exchange kinetics with the substrates malate and citrate, indicating that the main interactions between the proteins and di- and tricarboxylate substrates were not altered. In contrast, the interaction of the proteins with the monocarboxylate substrates mandelate and 2-hydroxyisovalerate changed in a complementary manner. The affinity of CitP for the S-enantiomers of these substrates was at least 1 order of magnitude lower than observed for MleP. Introduction of the C-terminal residues of MleP in CitP resulted in a higher affinity and vice versa. Interchanging the C-termini had a more complicated effect on the R-enantiomers, affecting different kinetic parameters with different substrates, indicating multiple interactions of the R groups at this side of the binding pocket. It is suggested that the binding pocket is located between transmembrane segment XI and the other transmembrane segments of the transporters

Transmembrane segment (TMS) VIII of the Na+/citrate transporter CitS requires downstream TMS IX for insertion in the Escherichia coli membrane

The amino acid sequence of the sodium ion-dependent citrate transporter CitS of IL pneumoniae contains 12 hydrophobic stretches that could form membrane-spanning segments. A previous analysis of the membrane topology in Escherichia coli using the PhoA gene fusion technique indicated that only nine of these hydrophobic segments span the membrane, while three segments, Vb, VIII and IX were predicted to have a periplasmic location (Van Geest, IM., and Lolkema, J. S. (1996) J. Biol. Chem. 271, 25582-25589), A topology study of C-terminally truncated CitS molecules in dog pancreas microsomes revealed that the protein traverses the endoplasmic reticulum membrane 11 times. In agreement with the PhoA fusion data, segment Vb was predicted to have a periplasmic location, but, in contrast, segments VIII and IX were found to be membrane-spanning (Van Geest, M., Nilsson, I., von Heijne, G., and Lolkema, J, S, (1999) J. Biol, Chem. 274, 2816-2823),In the present study, using site-directed Cys labeling, the topology of segments VIII and IX in the fall-length CitS protein was determined in the E. coli membrane, Engineered cysteine residues in the loop between the two segments were accessible to a membrane-impermeable thiol reagent exclusively from the cytoplasmic side of the membrane, demonstrating that transmembrane segments (TMSs) VIII and M are both membrane-spanning. It follows that the folding of CitS in the E. cold and endoplasmic reticulum membrane is the same. Cysteine accessibility studies of CitS-PhoA fusion molecules dem; onstrated that in the E. coli membrane segment VIII is exported to the periplasm in the absence of the C-terminal CitS sequences, thus explaining why the PhoA fusions do not correctly predict the topology. An engineered cysteine residue downstream of TMS VIII moved from a periplasmic to a cytoplasmic location when the fusion protein containing TMSs I-VIII was extended with segment IX, Thus, downstream segment M is both essential and sufficient for the insertion of segment VIII of CitS in the E. coli membrane.</p