Alternative-substrate inhibition and the kinetic mechanism of the beta-galactoside/proton symport of Escherichia coli.
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ABSTRACT:
SUBMITTER: Page MG
PROVIDER: S-EPMC1158407 | biostudies-other | 1982 Jun
REPOSITORIES: biostudies-other
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Project description:beta-Galactoside transport by Escherichia coli occurs with the concomitant uptake of a proton. The kinetics of beta-galactoside uptake at various values of external pH are interpreted in terms of a model in which both the galactoside and the proton are substrates of the transport reaction. The values of some of the kinetic constants for this two-substrate reaction were determined. The observed effects of the protonmotive force on the apparent Michaelis constant for galactoside can be explained in terms of the proton being a substrate of the transport reaction.
| S-EPMC1163091 | biostudies-other
Project description:The kinetics of galactoside-proton symport catalysed by a wild-type strain and one carrying a mutation, previously reported to cause uncoupling of the symport reaction, have been examined. The mutation does not affect the stoichiometry during the initial period of uptake, when the internal concentration of galactoside is low, but it does result in much greater competition from the galactoside as it is accumulated. Simple methods for the analysis of the uptake progress curves have been developed and used to estimate the initial rate of uptake and affinity for internal galactoside. The maximum rate of uptake is decreased by a factor of 2 at most whereas the affinity for internal galactoside is increased up to 50-fold by the mutation. The pH-dependence of the galactoside efflux reaction is changed in a manner which suggests that the defect is in the interaction between proton-binding and galactoside-binding sites rather than in the structure of either site.
| S-EPMC1147739 | biostudies-other
Project description:LacY catalyzes accumulation of galactosides against a concentration gradient by coupling galactoside and H+ transport (i.e., symport). While alternating access of sugar- and H+-binding sites to either side of the membrane is driven by binding and dissociation of sugar, the electrochemical H+ gradient ([Formula: see text]) functions kinetically by decreasing the Km for influx 50- to 100-fold with no change in Kd The affinity of protonated LacY for sugar has an apparent pK (pKapp) of ∼10.5, due specifically to the pKa of Glu325, a residue that plays an irreplaceable role in coupling. In this study, rates of lactose/H+ efflux were measured from pH 5.0 to 9.0 in the absence or presence of a membrane potential (ΔΨ, interior positive), and the effect of the imposed ΔΨ on the kinetics of efflux was also studied in right-side-out membrane vesicles. The findings reveal that [Formula: see text] induces an asymmetry in the transport cycle based on the following observations: 1) the efflux rate of WT LacY exhibits a pKapp of ∼7.2 that is unaffected by the imposed ΔΨ; 2) ΔΨ increases the rate of efflux at all tested pH values, but enhancement is almost 2 orders of magnitude less than observed for influx; 3) mutant Glu325 - Ala does little or no efflux in the absence or presence of ΔΨ, and ambient pH has no effect; and 4) the effect of ΔΨ (interior positive) on the Km for efflux is almost insignificant relative to the 50- to 100-fold decrease in the Km for influx driven by ΔΨ (interior negative).
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Project description:The toxin produced by Bacillus anthracis, the causative agent of anthrax, is composed of three proteins: a translocase heptameric channel, (PA(63))(7), formed from protective antigen (PA), which allows the other two proteins, lethal and edema factors (LF and EF), to translocate across a host cell's endosomal membrane, disrupting cellular homeostasis. It has been shown that (PA(63))(7) incorporated into planar phospholipid bilayer membranes forms a channel capable of transporting LF and EF. Protein translocation through the channel is driven by a proton electrochemical potential gradient on a time scale of seconds. A paradoxical aspect of this is that although LF(N) (the N-terminal 263 residues of LF), on which most of our experiments were performed, has a net negative charge, it is driven through the channel by a cis-positive voltage. We have explained this by claiming that the (PA(63))(7) channel strongly disfavors the entry of negatively charged residues on proteins to be translocated, and hence the aspartates and glutamates on LF(N) enter protonated (i.e., neutralized). Therefore, the translocated species is positively charged. Upon exiting the channel, the protons that were picked up from the cis solution are released into the trans solution, thereby making this a proton-protein symporter. Here, we provide further evidence of such a mechanism by showing that if only one SO(3)(-), which is essentially not titratable, is introduced at most positions in LF(N), through the reaction of an introduced cysteine residue at those positions with 2-sulfonato-ethyl-methanethiosulfonate, voltage-driven LF(N) translocation is drastically inhibited. We also find that a site that disfavors the entry of negatively charged residues into the (PA(63))(7) channel resides at or near its Phi-clamp, the ring of seven phenylalanines near the channel's entrance.
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Project description:The exit of lactose and thiomethyl-beta-D-galactoside from Escherichia coli ML308-225 has been studied to determine the role of carrier-dependent (zero-trans efflux) and carrier-independent (leak) processes. On the basis of its sensitivity to p-chloromercuribenzene sulphonate the exit of lactose was found to be almost wholly mediated by the carrier. Consistent with this conclusion was the finding that the rate of exit of this sugar was dependent on the external pH, being considerably slower at acid pH. On the other hand exit of thiomethyl-beta-D-galactoside was found to be composed of both carrier-dependent and carrier-independent processes. Both processes exhibited first-order kinetics with the rate constants for zero-trans efflux and leak being 0.137 min-1 and 0.079 min-1, respectively. The relevance of these findings for out earlier proposal for the methods of attenuation of solute accumulation is discussed [Booth, Mitchell & Hamilton (1979) Biochem. J. 182, 687--696].
| S-EPMC1161890 | biostudies-other
Project description:The aminoglycoside 3-N-acetyltransferase AAC(3)-IV from Escherichia coli exhibits a very broad aminoglycoside specificity, causing resistance to a large number of aminoglycosides, including the atypical veterinary antibiotic, apramycin. We report here on the characterization of the substrate specificity and kinetic mechanism of the acetyl transfer reaction catalyzed by AAC(3)-IV. The steady-state kinetic parameters revealed a narrow specificity for the acyl-donor and broad range of activity for aminoglycosides. AAC(3)-IV has the broadest substrate specificity of all AAC(3)'s studied to date. Dead-end inhibition and ITC experiments revealed that AAC(3)-IV follows a sequential, random bi-bi kinetic mechanism. The analysis of the pH dependence of the kinetic parameters revealed acid- and base-assisted catalysis and the existence of three additional ionizable groups involved in substrate binding. The magnitude of the solvent kinetic isotope effects suggests that a chemical step is at least partially rate limiting in the overall reaction.
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Project description:Inhibition of siderophore biosynthetic pathways in pathogenic bacteria represents a promising strategy for antibacterial drug development. Escherichia coli synthesize and secrete the small molecule iron chelator siderophore, enterobactin, in response to intracellular iron depletion. Here we describe a detailed kinetic analysis of EntE, one of six enzymes in the enterobactin synthetase gene cluster. EntE catalyzes the ATP-dependent condensation of 2,3-dihydroxybenzoic acid (DHB) and phosphopantetheinylated EntB (holo-EntB) to form covalently arylated EntB, a product that is vital for the final assembly of enterobactin. Initial velocity studies show that EntE proceeds via a bi-uni-uni-bi ping-pong kinetic mechanism with a k(cat) equal to 2.8 s(-1) and K(m) values of 2.5, 430, and 2.9 microM for DHB, ATP, and holo-EntB-ArCP, respectively. Inhibition and direct binding experiments suggest that, during the first half-reaction (adenylation), DHB binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), phosphopantetheinylated EntB binds to the enzyme followed by the release of products, AMP and arylated EntB. Two hydrolytically stable adenylate analogues, 5'-O-[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) and 5'-O-[N-(2,3-dihydroxybenzoyl)sulfamoyl]adenosine (DHB-AMS), are shown to act as slow-onset tight-binding inhibitors of the enzyme with (app)K(i) values of 0.9 and 3.8 nM, respectively. Direct binding experiments, via isothermal titration calorimetry, reveal low picomolar dissociation constants for both analogues with respect to EntE. The tight binding of Sal-AMS and DHB-AMS to EntE suggests that these compounds may be developed further as effective antibiotics targeted to this enzyme.
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Project description:Escherichia coli NhaA is a prototypical sodium-proton antiporter responsible for maintaining cellular ion and volume homeostasis by exchanging two protons for one sodium ion; despite two decades of research, the transport mechanism of NhaA remains poorly understood. Recent crystal structure and computational studies suggested Lys300 as a second proton-binding site; however, functional measurements of several K300 mutants demonstrated electrogenic transport, thereby casting doubt on the role of Lys300. To address the controversy, we carried out state-of-the-art continuous constant pH molecular dynamics simulations of NhaA mutants K300A, K300R, K300Q/D163N, and K300Q/D163N/D133A. Simulations suggested that K300 mutants maintain the electrogenic transport by utilizing an alternative proton-binding residue Asp133. Surprisingly, while Asp133 is solely responsible for binding the second proton in K300R, Asp133 and Asp163 jointly bind the second proton in K300A, and Asp133 and Asp164 jointly bind two protons in K300Q/D163N. Intriguingly, the coupling between Asp133 and Asp163 or Asp164 is enabled through the proton-coupled hydrogen-bonding network at the flexible intersection of two disrupted helices. These data resolve the controversy and highlight the intricacy of the compensatory transport mechanism of NhaA mutants. Alternative proton-binding site and proton sharing between distant aspartates may represent important general mechanisms of proton-coupled transport in secondary active transporters.
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Project description:Proton-dependent oligopeptide transporters (POTs) are major facilitator superfamily (MFS) proteins that mediate the uptake of peptides and peptide-like molecules, using the inwardly directed H(+) gradient across the membrane. The human POT family transporter peptide transporter 1 is present in the brush border membrane of the small intestine and is involved in the uptake of nutrient peptides and drug molecules such as ?-lactam antibiotics. Although previous studies have provided insight into the overall structure of the POT family transporters, the question of how transport is coupled to both peptide and H(+) binding remains unanswered. Here we report the high-resolution crystal structures of a bacterial POT family transporter, including its complex with a dipeptide analog, alafosfalin. These structures revealed the key mechanistic and functional roles for a conserved glutamate residue (Glu310) in the peptide binding site. Integrated structural, biochemical, and computational analyses suggested a mechanism for H(+)-coupled peptide symport in which protonated Glu310 first binds the carboxyl group of the peptide substrate. The deprotonation of Glu310 in the inward open state triggers the release of the bound peptide toward the intracellular space and salt bridge formation between Glu310 and Arg43 to induce the state transition to the occluded conformation.
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Project description:Substrate inhibition, whereby enzymatic activity decreases with excess substrate after reaching a maximum turnover rate, is among the most elusive phenomena in enzymatic catalysis. Here, based on a dynamic energy landscape model, we investigate the underlying mechanism by performing molecular simulations and frustration analysis for a model enzyme adenylate kinase (AdK), which catalyzes the phosphoryl transfer reaction ATP + AMP ⇋ ADP + ADP. Intriguingly, these reveal a kinetic repartitioning mechanism of substrate inhibition, whereby excess substrate AMP suppresses the population of an energetically frustrated, but kinetically activated, catalytic pathway going through a substrate (ATP)-product (ADP) cobound complex with steric incompatibility. Such a frustrated pathway plays a crucial role in facilitating the bottleneck product ADP release, and its suppression by excess substrate AMP leads to a slow down of product release and overall turnover. The simulation results directly demonstrate that substrate inhibition arises from the rate-limiting product-release step, instead of the steps for populating the catalytically competent complex as often suggested in previous works. Furthermore, there is a tight interplay between the enzyme conformational equilibrium and the extent of substrate inhibition. Mutations biasing to more closed conformations tend to enhance substrate inhibition. We also characterized the key features of single-molecule enzyme kinetics with substrate inhibition effect. We propose that the above molecular mechanism of substrate inhibition may be relevant to other multisubstrate enzymes in which product release is the bottleneck step.
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