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PubMed2003

Proton pump inhibitors in children: a review.

Proton pump inhibitors are often used to treat disorders associated with gastric hypersecretion in children, despite the lack of pediatric formulations. They are highly effective in the treatment of ulcers, gastro-esophageal reflux disorders and hypersecretory diseases. They provide a high level of gastric acid inhibition with few adverse effects. The aim of this article is to review the available studies concerning the use of proton pump inhibitors in pediatric populations and to point out: indications for use in children, optimal dosage, risk of adverse effects and consequences of the mechanism of action, and drug interactions. We performed a Medline and Embase search of publications printed from January 1980 to December 2002 concerning the use of proton pump inhibitors in children. We consider the available randomised controlled trials and several other uncontrolled studies conducted in the pediatric population, including all available information concerning the pediatric use of proton pump inhibitors. In children as well as in adults, there are clinical conditions (i.e., severe esophagitis or eradication of Helicobacter pylori) in which proton pump inhibitors offer clear advantages over histamine-2 receptor antagonists. The relatively common use of acid inhibitors (proton pump inhibitors and histamine-2 receptor antagonists) in uncomplicated gastro-esophageal reflux disorders or in the prevention of non-steroidal anti-inflammatory drugs/steroid gastropathy is often unsubstantiated and should be limited to very specific situations. Multicentre randomised controlled studies are needed to better define the efficacy profile, the optimal dosage with respect to the different indications and the safety profile for chronic therapy of proton pump inhibitors in children.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2005

Electron transfer ion/ion reactions in a three-dimensional quadrupole ion trap: reactions of doubly and triply protonated peptides with SO2*-.

Ion-ion reactions between a variety of peptide cations (doubly and triply charged) and SO2 anions have been studied in a 3-D quadrupole ion trap, resulting in proton and electron transfer. Electron transfer dissociation (ETD) gives many c- and z-type fragments, resulting in extensive sequence coverage in the case of triply protonated peptides with SO2*-. For triply charged neurotensin, in which a direct comparison can be made between 3-D and linear ion trap results, abundances of ETD fragments relative to one another appear to be similar. Reactions of doubly protonated peptides with SO2*- give much less structural information from ETD than triply protonated peptides. Collision-induced dissociation (CID) of singly charged ions formed in reactions with SO2*- shows a combination of proton and electron transfer products. CID of the singly charged species gives more structural information than ETD of the doubly protonated peptide, but not as much information as ETD of the triply protonated peptide.

Electron Transport↗
PubMed1995

Pathway of proton transfer in bacterial reaction centers: further investigations on the role of Ser-L223 studied by site-directed mutagenesis.

The role of Ser-L223 in proton transfer to reduced QB in the reaction center (RC) from Rhodobacter sphaeroides was studied by site-directed replacement of Ser with residues having different proton donor properties, e.g., the aliphatic residues Ala and Gly, the hydroxyl residue Thr, the amide residue Asn, the sulhydryl residue Cys, the imidazole residue His, and the carboxylic acid residue Asp. Compared to native reaction centers, RCs with Ala or Asn at L223 had greatly reduced (approximately 300-fold) proton-coupled electron transfer rates, kAB(2), associated with the second electron reduction of QB (QA(-)QB(-) + H+ --> QAQBH-). In contrast, RCs containing Thr, Asp, or Gly at L223 retained fast proton-coupled electron transfer rates. RCs with His or Cys at L223 did not bind the secondary quinone QB. These results show that kAB(2) is larger when a good proton transfer group, e.g., a hydroxyl residue (Ser, Thr) or a carboxylic acid (Asp), occupies the L223 site, supporting the proposal that Ser-L223 is a component of a proton transfer chain [Paddock, M. L., McPherson, P. H., Feher, G., & Okamura, M. Y. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6803--6807]. The surprising result that kAB(2) is not significantly reduced in RCs with Gly at L223 suggests that a water molecule functionally replaces the missing Ser hydroxyl group in the mutant RCs. The importance of Ser-L223 in internal proton transfer reactions within the RC is discussed.

Base Sequence↗
PubMed1993

Relationship of proton uptake on the cytoplasmic surface and reisomerization of the retinal in the bacteriorhodopsin photocycle: an attempt to understand the complex kinetics of the pH changes and the N and O intermediates.

In the bacteriorhodopsin photocycle the recovery of the initial BR state from the M intermediate occurs via the N and O intermediates. The molecular events in this process include reprotonation of the Schiff base and the subsequent uptake of a proton from the cytoplasmic side, as well as reisomerization of the retinal from 13-cis to all-trans. We have studied the kinetics of the intermediates and the proton uptake. At moderately low pH little of the N state accumulates, and the O state dominates in the reactions that lead from M to BR. The proton uptake lags behind the formation of O, suggesting the sequence N(0)<==>O(0) + H+ (from the bulk)-->O(+1)-->BR+H+ (to the bulk), where the superscripts indicate the net protonation state of the protein relative to BR. Together with a parallel study of ours at moderately high pH, these results suggest that the sequence of proton uptake and retinal reisomerization depends on pH: at low pH the isomerization occurs first and O accumulates, but at high pH the isomerization is delayed and therefore N accumulates. Although this model contains too many rate constants for rigorous testing, we find that it will generate most of the characteristic pH-dependent kinetic features of the photocycle with few assumptions other than pH dependency for protonation at the proton release and uptake steps.

Amino Acid Sequence↗
PubMed1993

Rate-equilibria relationships in intramolecular proton transfer in human carbonic anhydrase III.

Maximal turnover rates for the dehydration of HCO3- catalyzed by the zinc metalloenzyme carbonic anhydrase III are limited by a proton transfer to zinc-bound hydroxide in the active site. We have used site-directed mutagenesis to place a proton donor, histidine, at position 64 and used 18O exchange between CO2 and water measured by mass spectrometry to determine the rates of intramolecular proton transfer to the zinc-bound hydroxide. In a series of site-specific mutants, the values of pKa of the zinc-bound water ranged from approximately 5 to 9. The rate constants for proton transfer obeyed a Brønsted correlation and showed sharp curvature characteristic of facile proton transfers. Application of Marcus rate theory shows that this proton transfer has the small intrinsic energy barrier (near 1.5 kcal/mol) characteristic of rapid proton transfer between nitrogen and oxygen acids and bases, but has an observed overall energy barrier (near 10 kcal/mol), indicating the involvement of accompanying, energy requiring processes such as solvent reorganization or conformational change.

Amino Acid Sequence↗
PubMed1983

Nuclear magnetic resonance study on the exchange behavior of the NH-N protons of a ribonucleic acid miniduplex.

The exchange behavior of the guanine N(1) and uracil N(3) protons in the self-complementary hexanucleotide r(ApApGpCpUpU) has been studied at 5 degrees C in 80% H2O/20% D2O by proton NMR. Under these conditions, the hexanucleotide forms a stable miniduplex. The exchange rate of all Watson-Crick NH protons is unaffected by addition of trifluoroethylamine up to 0.07 M. On the other hand, addition of phosphate buffer, pH 6.9, enhances the exchange rate of the uracil N(3) protons of both terminal and internal A X U base pairs but does not influence the exchange rate of the guanine N(1) protons of the central G X C base pairs. Catalysis by increased phosphate concentrations results in an open-limited rate of the internal A X U base pairs with kex = 233 s-1, equivalent to a lifetime of 4.3 ms. The proton exchange of the central G X C is regulated by the opening rate of the central core of the miniduplex. On the other hand, the sensitivity of the exchange rate of internal as well as of terminal A X U base pairs can be explained by their reduced lifetime due to end "fraying" and a subsequent catalysis of the exchange process from the opened state. These results suggest that it may be possible to probe labilized parts of RNAs such as tRNA by gradual addition of the exchange catalyst phosphate and to monitor their exchange rates by proton NMR.

Base Composition↗
PubMed1983

Exceptional characteristics of amino proton exchange in guanosine compounds.

Amino 1H NMR line width as a measure of amino proton exchange in guanosine compounds is completely unaffected by the addition of ca. 1 M tris(hydroxymethyl)-aminomethane, imidazole, 2-(N-morpholino)ethanesulfonic acid, glycine, or cacodylate, all shown to be effective buffer catalysts in adenosine and cytidine proton exchange. Line broadening, seen only with phosphate and acetate, is established by intermolecular interactions, as well as by amino to water proton exchange. This absence of buffer catalysis of exchange is accounted for by the relatively small implied effect of G(N-7) protonation on amino acidity, based on similar observations with 7-methylguanosine as a model for endocyclic protonation. The requirement for diffusion-controlled proton transfer in buffer catalysis is achieved by nucleobase protonation in adenine and cytosine, but not in guanine.

Amines↗
PubMed1989

pH dependence of the kinetic parameters for the pyrophosphate-dependent phosphofructokinase reaction supports a proton-shuttle mechanism.

The pH dependence of kinetic parameters for the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-1 of F6P and then donates it to protonate the leaving phosphate. Whether this occurs in one or two steps cannot be deduced from the present data. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when correctly protonated and only to the correctly protonated form of the enzyme. Specifically, the requirement for two enzyme epsilon-amino groups in the protonated form for reactant binding was detected as was the requirement for the ionized phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, MgPPi and HPO4(2-). The protonation state of enzyme and reactant binding groups is in agreement with data obtained previously [Cho, Y.-K., & Cook, P. F. (1988) J. Biol. Chem. 263, 5135].

Binding Sites↗
PubMed1990

Proton diffusion in the active site of triosephosphate isomerase.

The current model for hydrogen flow in the aldose-ketose isomerases is probably incorrect. Enzymes of this class are characterized by both hydrogen transfer and proton exchange in the interconversion of substrate and product. The transfer is believed to be due to the action of a unique basic residue in the active site. Exchange is presumed to occur by dissociation of the abstracted proton and reassociation from the medium prior to its transfer to the intermediate enediol on the way to product. Dissociation of a necessary proton from the intermediate state imposes limits on the overall catalytic rate depending on the pKa of the protonated base and the pH of the medium. A case in point is triose-P isomerase (TIM), where kcat is approximately 10(4) s-1. T-Labeled substrate is found to lose approximately 95% of its T to the medium when totally converted to product. Although the active site base is believed to be a glutamate of pKa = 3.9, the pH dependence of maximum velocity is known to be flat up to pH 10. The loss of hydrogen required to form product as indicated by isotope exchange must be restored completely at this high pH, requiring a base of very high pKa, or there must be some other explanation for the loss of isotope. The present study demonstrates the existence of a single proton on human and rabbit TIM and three protons on yeast TIM that rapidly exchange with the abstracted proton at the E.enediol state internal exchange. Exchange with the medium external exchange occurs from the enzyme after substrate or product has dissociated.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2001

Simultaneous replacement of Asp-L210 and Asp-M17 with Asn increases proton uptake by Glu-L212 upon first electron transfer to QB in reaction centers from Rhodobacter sphaeroides.

In the photosynthetic reaction center (RC) from Rhodobacter sphaeroides, the first electron transfer to the secondary quinone acceptor Q(B) is coupled to the protonation of Glu-L212, located approximately 5 A from the center of Q(B). Upon the second electron transfer to Q(B), Glu-L212 is involved in fast proton delivery to the reduced Q(B). Since Asp-L210 and Asp-M17 play an important role in the proton transfer to the Q(B) site [Paddock, M. L., Adelroth, P., Chang, C., Abresch, E. C., Feher, G., and Okamura, M. Y. (2001) Biochemistry 40, 6893-6902], we investigated the effects of replacing one or both Asp residues with Asn on proton uptake by Glu-L212 using FTIR difference spectroscopy. Upon the first electron transfer to Q(B), the amplitude of the proton uptake by Glu-L212 at pH 8 is increased in the single and double mutant RCs, as is evident from the larger intensity (by 35-55%) of the carboxylic acid band at 1727 cm(-1) in the Q(B)(-)/Q(B) difference spectra of mutant RCs, compared to that at 1728 cm(-1) in native RCs. This implies that the extent of ionization of Glu-L212 in the Q(B) ground state is greater in the mutants than in native RCs and that Asp-M17 and Asp-L210 are at least partially ionized near neutral pH in native RCs. In addition, no changes in the protonation state or the environment of these two residues are detected upon Q(B) reduction. The absence of the 1727 cm(-1) signal in all of the RCs lacking Glu-L212, confirms that the positive band at 1728-1727 cm(-1) probes the protonation of Glu-L212 in native and mutant RCs.

Amino Acid Substitution↗
PubMed2002

The entry point of the K-proton-transfer pathway in cytochrome c oxidase.

Cytochrome c oxidase is a redox-driven proton pump. The enzyme has two proton input pathways, leading from the solution on the N-side to the binuclear center. One of these pathways, the K-pathway, is used for proton uptake upon reduction of the binuclear center. It is also important for local charge compensation during reaction of the fully reduced enzyme with O2. Two different locations have been proposed to constitute the entry point of the K-pathway: near S(I-299) or near E(II-101), respectively, in the Rhodobacter sphaeroides enzyme. The experiments discussed in this study are aimed at identifying the location of the entry point. The kinetics and extent of flash-induced proton release coupled to oxidation of heme a3 (tau congruent with 2 ms at pH 8.8 in the wild-type enzyme) in the absence of O2 were investigated in the ED(II-101), SD(I-299), and KM(I-362) mutant enzymes, i.e., at the two proposed entry points and in the middle of the pathway, respectively. This reaction was completely blocked in KM(I-362), while it was slowed by factors of 25 and 40 in the ED(II-101) and SD(I-299) mutant enzymes, respectively. During reaction of the fully reduced enzyme with O2, electron transfer from heme a to the catalytic site (during P(R)-formation) was blocked in the KM(I-362) and SD(I-299)/SG(I-299) but not in the ED(II-101)/ EA(II-101) mutant enzymes. The results are interpreted as follows: Residue K(I-362) is involved in both proton transfer and charge compensation (in different reaction steps). The impaired proton release in the S(I-299) mutant enzymes is an indirect effect due to an altered environment of K(I-362). E(II-101), on the other hand, is likely to be part of the K-pathway since mutation of this residue results in impaired proton release but does not affect the P(R) formation kinetics; i.e., the properties of K(I-362) are not altered. Consequently, we conclude that the entry point of the K-pathway is located near E(II-101).

Carbon Monoxide↗
PubMed2003

Calculation of proton transfers in Bacteriorhodopsin bR and M intermediates.

Residue ionization states were calculated in nine crystal structures of bacteriorhodopsin trapped in bR, early M, and late M states by multiconformation continuum electrostatics. This combines continuum electrostatics and molecular mechanics, deriving equilibrium distributions of ionization states and polar residue and water positions. The three central cluster groups [retinal Schiff base (SB), Asp 85 and Asp 212] are ionized in bR structures while a proton has transferred from SB(+) to Asp 85(-) in late M structures matching experimental results. The proton shift in M is due to weaker SB(+)-ionized acid and more favorable SB(0)-ionized acid interactions following retinal isomerization. The proton release cluster (Glu 194 and Glu 204) binds one proton in bR, which is lost to water by pH 8 in late M. In bR the half-ionized state is stabilized by charge-dipole interactions while full ionization is disallowed by charge-charge repulsion between the closely spaced acids. In M the acids move apart, permitting full ionization. Arg 82 movement connects the proton shifts in the central and proton release clusters. Changes in total charge of the two clusters are coupled by direct long-range interactions. Separate calculations consider continuum or explicit water in internal cavities. The explicit waters and nearby polar residues can reorient to stabilize different charge distributions. Proton release to the low-pH, extracellular side of the protein occurs in these calculations where residue ionization remains at equilibrium with the medium. Thus, the key changes distinguishing the intermediates are indeed trapped in the structures.

Aspartic Acid↗
PubMed2003

Mechanism of proton transfer inhibition by Cd(2+) binding to bacterial reaction centers: determination of the pK(A) of functionally important histidine residues.

The bacterial photosynthetic reaction center (RC) uses light energy to catalyze the reduction of a bound quinone molecule Q(B) to quinol Q(B)H(2). In RCs from Rhodobacter sphaeroides the protons involved in this process come from the cytoplasm and travel through pathways that involve His-H126 and His-H128 located near the proton entry point. In this study, we measured the pH dependence from 4.5 to 8.5 of the binding of the proton transfer inhibitor Cd(2+), which ligates to these surface His in the RC and inhibits proton-coupled electron transfer. At pH <6, the negative slope of the logarithm of the dissociation constant, K(D), versus pH approaches 2, indicating that, upon binding of Cd(2+), two protons are displaced; i.e., the binding is electrostatically compensated. At pH >7, K(D) becomes essentially independent of pH. A theoretical fit to the data over the entire pH range required two protons with pK(A) values of 6.8 and 6.3 (+/-0.5). To assess the contribution of His-H126 and His-H128 to the observed pH dependence, K(D) was measured in mutant RCs that lack the imidazole group of His-H126 or His-H128 (His --> Ala). In both mutant RCs, K(D) was approximately pH independent, showing that Cd(2+) does not displace protons upon binding in the mutant RCs, in contrast to the native RC in which His-H126 and His-H128 are the predominant contributors to the observed pH dependence of K(D). Thus, Cd(2+) inhibits RC function by binding to functionally important histidines.

Aspartic Acid↗
PubMed2005

Structural and kinetic characterization of active-site histidine as a proton shuttle in catalysis by human carbonic anhydrase II.

In the catalysis of the hydration of carbon dioxide and dehydration of bicarbonate by human carbonic anhydrase II (HCA II), a histidine residue (His64) shuttles protons between the zinc-bound solvent molecule and the bulk solution. To evaluate the effect of the position of the shuttle histidine and pH on proton shuttling, we have examined the catalysis and crystal structures of wild-type HCA II and two double mutants: H64A/N62H and H64A/N67H HCA II. His62 and His67 both have their side chains extending into the active-site cavity with distances from the zinc approximately equivalent to that of His64. Crystal structures were determined at pH 5.1-10.0, and the catalysis of the exchange of (18)O between CO(2) and water was assessed by mass spectrometry. Efficient proton shuttle exceeding a rate of 10(5) s(-)(1) was observed for histidine at positions 64 and 67; in contrast, relatively inefficient proton transfer at a rate near 10(3) s(-)(1) was observed for His62. The observation, in the crystal structures, of a completed hydrogen-bonded water chain between the histidine shuttle residue and the zinc-bound solvent does not appear to be required for efficient proton transfer. The data suggest that the number of intervening water molecules between the donor and acceptor supporting efficient proton transfer in HCA II is important, and furthermore suggest that a water bridge consisting of two intervening water molecules is consistent with efficient proton transfer.

Alanine↗
PubMed2004

Identification of a novel protonation pattern for carboxylic acids upon Q(B) photoreduction in Rhodobacter sphaeroides reaction center mutants at Asp-L213 and Glu-L212 sites.

In the reaction center from the photosynthetic purple bacterium Rhodobacter sphaeroides, light energy is rapidly converted to chemical energy through coupled electron-proton transfer to a buried quinone molecule Q(B). Involved in the proton uptake steps are carboxylic acids, which have characteristic infrared vibrations that are observable using light-induced Fourier transform infrared (FTIR) difference spectroscopy. Upon formation, Q(B)(-) induces protonation of Glu-L212, located within 5 A of Q(B), resulting in a IR signal at 1728 cm(-1). However, no other IR signal is observed within the classic absorption range of protonated carboxylic acids (1770-1700 cm(-1)). In particular, no signal for Asp-L213 is found despite its juxtaposition to Q(B) and importance for proton uptake on the second electron-transfer step. In an attempt to uncover the reason behind this lack of signal, the microscopic electrostatic environment in the vicinity of Q(B) was modified by interchanging Asp and Glu at the L213 and L212 positions. The Q(B)(-)/Q(B) FTIR spectrum of the Asp-L212/Glu-L213 swap mutant in the 1770-1700 cm(-1) range shows several distinct new signals, which are sensitive to (1)H/(2)H isotopic exchange, indicating that the reduction of Q(B) results in the change of the protonation state of several carboxylic acids. The new bands at 1752 and 1747 cm(-1) were assigned to an increase of protonation in response to Q(B) reduction of Glu-L213 and Asp-L212, respectively, based on the effect of replacing them with their amine analogues. Since other carboxylic acid signals were observed, it is concluded that the swap mutations at L212 and L213 affect a cluster of carboxylic acids larger than the L212/L213 acid pair. Implications for the native reaction center are discussed.

Amino Acid Substitution↗
PubMed2005

An arginine to lysine mutation in the vicinity of the heme propionates affects the redox potentials of the hemes and associated electron and proton transfer in cytochrome c oxidase.

Cytochrome c oxidase pumps protons across a membrane using energy from electron transfer and reduction of oxygen to water. It is postulated that an element of the energy transduction mechanism is the movement of protons to the vicinity of the hemes upon reduction, to favor charge neutrality. Possible sites on which protons could reside, in addition to the conserved carboxylate E286, are the propionate groups of heme a and/or heme a(3). A highly conserved pair of arginines (R481 and R482) interact with these propionates through ionic and hydrogen bonds. This study shows that the conservative mutant, R481K, although as fully active as the wild type under many conditions, exhibits a significant decrease in the midpoint redox potential of heme a relative to Cu(A) (DeltaE(m)) of approximately equal 40 mV, has lowered activity under conditions of high pH or in the presence of a membrane potential, and has a slowed heme a(3) reduction with dithionite. Another mutant, D132A, which strongly inhibits proton uptake from the internal side of the membrane, has <4% of the activity of the wild type and appears to be dependent on proton uptake from the outside. A double mutation, D132A/R481K, is even more strongly inhibited ( approximately 1% of that of the wild type). The more-than-additive effect supports the concept that R481K not only lowers the midpoint potential of heme a but also limits a supply route for protons from the outside of the membrane used by the D132 mutant. The results are consistent with an important role of R481 and heme a/a(3) propionates in proton movement in a reversible exit path.

Animals↗
PubMed2006

Catalytic role of proton transfers in the formation of a tetrahedral adduct in a serine carboxyl peptidase.

Quantum mechanical/molecular mechanical molecular dynamics and 2D free energy simulations are performed to study the formation of a tetrahedral adduct by an inhibitor N-acetyl-isoleucyl-prolyl-phenylalaninal (AcIPF) in a serine-carboxyl peptidase (kumamolisin-As) and elucidate the role of proton transfers during the nucleophilic attack by the Ser278 catalytic residue. It is shown that although the serine-carboxyl peptidases have a fold resembling that of subtilisin, the proton transfer processes during the nucleophilic attack by the Ser residue are likely to be more complex for these enzymes compared to the case in classical serine proteases. The computer simulations demonstrate that both general base and acid catalysts are required for the formation and stabilization of the tetrahedral adduct. The 2D free energy maps further demonstrate that the proton transfer from Ser278 to Glu78 (the general base catalyst) is synchronous with the nucleophilic attack, whereas the proton transfer from Asp164 (the general acid catalyst) to the inhibitor is not. The dynamics of the protons at the active site in different stages of the nucleophilic attack as well as the motions of the corresponding functional groups are also studied. It is found that the side chain of Glu78 is generally rather flexible, consistent with its possible multifunctional role during catalysis. The effects of proton shuffling from Asp82 to Glu78 and from Glu32 to Asp82 are examined, and the results indicate that such proton shuffling may not play an important role in the stabilization of the tetrahedral intermediate analogue.

Aspartic Acid↗
PubMed1996

Glu46 donates a proton to the 4-hydroxycinnamate anion chromophore during the photocycle of photoactive yellow protein.

Photoactive yellow protein (PYP) is a photoreceptor containing a unique 4-hydroxycinnamic acid (pCA) chromophore. The trans to cis photoisomerization of this chromophore activates a photocycle involving first a short-lived red-shifted intermediate (pR), then a long-lived blue-shifted intermediate (pB), and finally recovery of the original receptor state (pG). The pCA chromophore is deprotonated in pG and protonated in pB, but the proton donor for this process has not yet been identified. Here we report the first FTIR spectroscopic data on pG, pR, and pB. The IR difference signals in the carbonyl stretching region of COOH groups (1700-1800 cm-1) reveal that a buried carboxylic group close to the chromophore (i) is protonated in pG, (ii) develops a stronger hydrogen bonding in pR, and (iii) becomes deprotonated in pB. These signals are unambiguously assigned to Glu46, on the basis of the IR data and the 1.4 A X-ray structure of PYP [Borgstahl et al. (1995) Biochemistry 34, 6278-6287]. Our data demonstrate that in pR Glu46 remains in hydrogen bonding contact with the negatively charged phenolic oxygen of pCA after chromophore photoisomerization. This strongly implies that the chromophore is isomerized to the 7-cis 9-s-trans conformation in pR, resulting from co-isomerization of both the C7 = C8 and C9-C10 bonds. In the pR to pB transition, Glu46 becomes deprotonated, concomitant with chromophore protonation. Therefore, we conclude that Glu46 functions as the proton donor for the protonation of pCA during the PYP photocycle. We propose a molecular mechanism in which intramolecular proton transfer in PYP leads to global protein conformational changes involved in signal transduction.

Bacterial Proteins↗