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1.
High resolution proton nuclear magnetic resonance has been used to observe protons at the active site of chymotrypsin Aδ and at the same region of chymotrypsinogen A. A single resonance with the intensity of one proton is located in the low field region of the nuclear magnetic resonance spectrum. This resonance is observed in H2O solutions but not in 2H2O. On going from low to high pH the resonance titrates upfield 3 parts per million in both proteins and has a pK of 7.5. The titration can be prevented by alkylating His57 with either of two active site directed chloromethyl ketones. Using these data the proton resonance has been assigned to a proton in a hydrogen bond between His57 and Asp102. Further confirmation of this assignment lies in the observation of a similar resonance in this same low field region of the nuclear magnetic resonance spectrum of trypsin, trypsinogen, subtilisin BPN′ and α-lytic protease all of which have the Asp-His-Ser triad at their active sites.This proton resonance in chymotrypsin Aδ was used as a probe to monitor the charge state of the active site upon formation of a stable acyl-enzyme analogue N2(N-acetylalanyl)-N1benzoylcarbazoyl-chymotrypsin Aδ. In this derivative the His-Asp proton resonance titrates from the same low pH end point as in the native enzyme, ?18 parts per million, to a new high pH end point of ?14.4 parts per million (versus ?15.0 parts per million in the native enzyme). The difference of 0.6 parts per million in the high pH end points between the native and acyl enzyme is interpreted as supporting the suggestion that a hydrogen bond exists between Ser195 and His57 in the native enzyme and zymogen.We conclude from these studies that the charge relay system from Asp102 across His57 to Ser195 is intact in chymotrypsin Aδ and chymotrypsinogen A, and that, in the native enzyme, it slightly polarizes Ser195. 相似文献
2.
High resolution nuclear magnetic resonance study of the histidine--aspartate hydrogen bond in chymotrypsin and chymotrypsinogen 总被引:2,自引:0,他引:2
A high resolution proton nuclear magnetic resonance study of chymotrypsin Aδ and Chymotrypsinogen A in water has shown a single resonance at very low magnetic fields (− 18 to − 15 p.p.m. relative to dimethyl-silapentane-sulfonate). From its pH dependence (pK = 7·2) and response to chemical modification the resonance has been assigned to the hydrogen-bonded proton between His-57 and Asp-102. 相似文献
3.
Wahlgren WY Pál G Kardos J Porrogi P Szenthe B Patthy A Gráf L Katona G 《The Journal of biological chemistry》2011,286(5):3587-3596
The mechanism of serine proteases prominently illustrates how charged amino acid residues and proton transfer events facilitate enzyme catalysis. Here we present an ultrahigh resolution (0.93 Å) x-ray structure of a complex formed between trypsin and a canonical inhibitor acting through a substrate-like mechanism. The electron density indicates the protonation state of all catalytic residues where the catalytic histidine is, as expected, in its neutral state prior to the acylation step by the catalytic serine. The carboxyl group of the catalytic aspartate displays an asymmetric electron density so that the Oδ2–Cγ bond appears to be a double bond, with Oδ2 involved in a hydrogen bond to His-57 and Ser-214. Only when Asp-102 is protonated on Oδ1 atom could a density functional theory simulation reproduce the observed electron density. The presence of a putative hydrogen atom is also confirmed by a residual mFobs − DFcalc density above 2.5 σ next to Oδ1. As a possible functional role for the neutral aspartate in the active site, we propose that in the substrate-bound form, the neutral aspartate residue helps to keep the pKa of the histidine sufficiently low, in the active neutral form. When the histidine receives a proton during the catalytic cycle, the aspartate becomes simultaneously negatively charged, providing additional stabilization for the protonated histidine and indirectly to the tetrahedral intermediate. This novel proposal unifies the seemingly conflicting experimental observations, which were previously seen as either supporting the charge relay mechanism or the neutral pKa histidine theory. 相似文献
4.
At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated. 相似文献
5.
Katrin Bastyns Matheus Froeyen Jos Fernando Díaz Guido Volckaert Yves Engelborghs 《Proteins》1996,24(3):370-378
Barnase, the guanine specific ribonuclease of Bacillus amyloliquefaciens, was subjected to mutations in order to alter the electrostatic properties of the enzyme. Ser-85 was mutated into Glu with the goal to introduce an extra charge in the neighborhood of His-102. A double mutation (Ser-85-Glu and Asp-86-Asn) was introduced with the same purpose but without altering the global charge of the enzyme. A similar set of mutations was made using Asp at position 85. For all mutants the pI was determined using the technique of isoelectric focusing and calculated on the basis of the Tanford-Kirkwood theory. When Glu was used to replace Ser-85, the correlation between the experimental and the calculated values was perfect. However, in the Ser-85-Asp mutant, the experimental pI drop is bigger than the calculated one, and in the double mutant (Ser-85-Asp and Asp-86-Asn) the compensation is not achieved. The effect of the mutations on the pKa of His-102 can be determined from the pH dependence of the kcat/KM for the hydrolysis of dinucleotides, e.g., GpC. The effect can also be calculated using the method of Honig. In this case the agreement is very good for the Glu-mutants and the single Asp-mutant, but less for the double Asp-mutant. The global stability of the Asp-mutants is, however, the same as the wild type, as shown by stability studies using urea denaturation. Molecular dynamics calculations, however, show that in the double Asp-mutant His-102 (H+) swings out of its pocket to make a hydrogen bridge with Gln-104 which should cause an additional pKa rise. The effect of the Glu-mutations was also tested on all the kinetic parameters for GpC and the cyclic intermediate G > p at pH 6.5, for RNA at pH 8.0, and for poly(A) at pH 6.2. The effect of the mutations is rather limited for the dinucleotide and the cyclic intermediate, but a strong increase of the KM is observed in the case of the single mutant (extra negative charge) with polymeric substrates. These results indicate that the extra negative charge has a strong destabilizing effect on the binding of the polymeric substrates in the ground state and the transition state complex. A comparison with the structure of bound tetranucleotides (Buckle, A.M. and Fersht, A.R., Biochemistry 33:1644–1653, 1994) shows that the extra negative charge points towards the P2 site. 相似文献
6.
《BBA》1987,891(2):165-176
Bacteriorhodopsin and trypsin-modified bacteriorhodopsin have been reconstituted into liposomes by means of a low pH-sonication procedure. The incorporation of bacteriorhodopsin in these proteoliposomes is predominantly in the same direction as in vivo and the direction of proton pumping is from inside to outside the liposomes. The direction of proton translocation and electrical potential generation was studied as a function of the reconstitution pH. Light-dependent proton extrusion and generation of a Δp, interior negative and alkaline was observed at a reconstitution pH below 3.0 using bacteriorhodopsin, and at a pH below 3.5 using trypsin-modified bacteriorhodopsin. The shift in inflection point is explained in terms of differences between bacteriorhodopsin and trypsin-modified bacteriorhodopsin in a specific protein-phospholipid interaction which depends on the surface charge density of the cytoplasmic side of bacteriorhodopsin. The magnitude of the protonmotive force (Δp) generated by trypsin-modified bacteriorhodopsin in liposomes was quantitated. Illumination of the proteoliposomes resulted in the generation of a high Δp (135 mV, inside negative and alkaline), with a major contribution of the pH gradient. The ionophores nigericin and valinomycin induced, respectively, a compensatory interconversion of ΔpH into Δψ and vice versa. If no endogenous proton permeability of the membrane would exist, a protonmotive force could be generated of − 143 mV as electrical potential alone or − 162 mV as pH gradient alone. 相似文献
7.
The major proton-transfer pathway into the buried active site of cytochrome c oxidase (CcO) is the D-pathway that begins with the subunit I residue Asp-132 on the inner protein surface (the cytoplasmic surface of the aa3-type CcO of Rhodobacter sphaeroides). Asp-132 is surrounded by residues from both subunits I and III. In the absence of subunit III, CcO retains activity, but the functional characteristics of the D-pathway are significantly altered such that the transfer of protons from Asp-132 into the pathway becomes the rate-limiting step. Determination of the pH-dependence of the rate constant for D-pathway proton uptake during the single-turnover of CcO indicates that the pKa of Asp-132 in the absence of subunit III is approximately 7. The removal of subunit III also allows for alternative surface proton donor/acceptors other than Asp-132. With Asp-132 altered to alanine, the rate constant for D-pathway proton uptake is very slow (5 s(-1)) in the presence of subunit III. Once subunit III is removed, the proton uptake rate constant increases 80-fold, to 400 s(-1). The pKa associated with this uptake is >10, and the initial proton donor/acceptor in D132A III (-) is proposed to be a water of the D-pathway rather than an amino acid residue. Arachidonic acid (Aa), which stimulates the activity of several D-pathway mutant CcOs, appears to become the initial proton donor/acceptor in the absence of subunit III, whether or not Asp-132 is altered. Aa shifts the pKa of the initial proton donor to 7.6 for both wild-type (WT) III (-) and D132A III (-). The results indicate that subunit III creates a barrier that helps prevent protons from donors other than Asp-132 from directly accessing the internal waters of the D-pathway, while the subunit also provides an environment that increases the rate at which Asp-132 transfers protons into the D-pathway. 相似文献
8.
Sensory rhodopsin II (HsSRII, also called phoborhodopsin) is a negative phototaxis receptor of Halobacterium salinarum, a bacterium that avoids blue-green light. In this study, we expressed the protein in Escherichia coli cells, and reconstituted the purified protein with phosphatidylcholine. The reconstituted HsSRII was stable. We examined the photocycle by flash-photolysis spectroscopy in the time range of milliseconds to seconds, and measured proton uptake/release using a transparent indium-tin oxide electrode. The pKa of the counterion of the Schiff base, Asp73, was 3.0. Below pH 3, the depleted band was observed on flash illumination, but the positive band in the difference spectra was not found. Above pH 3, the basic photocycle was HsSRII (490) → M (350) → O (520) → Y (490) → HsSRII, where the numbers in parentheses are the maximum wavelengths. The decay rate of O-intermediate and Y-intermediate were pH-independent, whereas the M-intermediate decay was pH-dependent. For 3 < pH < 4.5, the M-decay was one phase, and the rate decreased with an increase in pH. For 4.5 < pH < 6.5, the decay was one phase with pH-independent rates, and azide markedly accelerated the M-decay. These findings suggest the existence of a protonated amino acid residue (X-H) that may serve as a proton relay to reprotonate the Schiff base. Above pH 6.5, the M-decay showed two phases. The fast M-decay was pH-independent and originated from the molecule having a protonated X-H, and the slow M-decay originated from the molecule having a deprotonated X, in which the proton came directly from the outside. The analysis yielded a value of 7.5 for the pKa of X-H. The proton uptake and release occurred during M-decay and O-decay, respectively. 相似文献
9.
The proton nuclear magnetic resonance signal of the His57-Asp102 hydrogen bonded proton in the charge relay system of chymotrypsinogen A and chymotrypsin Aδ has been monitored to determine the influence of substrate analogues and competitive inhibitors on the electronic state of the active site regions. Borate ion, benzene boronic acid and 2-phenylethylboronic acid, when bound to chymotrypsin at pH 9.5 shift the resonance position of the His-Asp hydrogen bonded proton to ?15.9, ?16.3 and ?17.2 parts per million, respectively. These positions are intermediate between the low pH position in the free enzyme of ?18.0 parts per million and the high pH position of ?14.9 parts per million. The presence of these analogues prevents the His-Asp proton resonance from titrating in the region of pH 6 to 9.5. Similar low field shifts are observed for the hydrogen bonded proton resonance of subtilisin BPN′ when complexed with these boronic acids. The results support the chemical and crystallographic data which show that negatively charged tetrahedral adducts of the boronic acid substrate analogues are formed at the active sites of these enzymes. When combined with similar nuclear magnetic resonance data for the binding of N-acetyl-l-tryptophan to chymotrypsin Aδ, they suggest that a direct interaction occurs between the active site histidine and the atom occupying the leaving group position of the substrate, presumably a hydrogen bond.The His-Asp proton resonance was also monitored in complexes of chymotrypsin Aδ with bovine pancreatic trypsin inhibitor over the pH range 4 to 9. In the complex the low field proton resonance had a field position of ?14.9 parts per million over the pH range 4 to 9 indicating that His57 is in the neutral form, similar to the active enzyme at high pH. 相似文献
10.
Christof Klughammer Katharina Siebke Ulrich Schreiber 《Photosynthesis research》2013,117(1-3):471-487
Technical features and examples of application of a special emitter–detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550–520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and CO2 uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal CO2 concentrations, except for very low CO2, where the rate of charge flux distinctly exceeded the rate of CO2 uptake. Parallel oscillations in CO2 uptake and charge flux were induced by high CO2 and O2. The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves. 相似文献
11.
The use of algae as a potential platform for fuels or biochemical production requires process design and control that can be implemented at agronomic scales. Toward achieving pH control in large unmixed systems, we present a rigorous set of direct measurements of non-buffered proton uptake and efflux during growth on ammonium and nitrate, observing nearly unit molar proton imbalance H+/OH? respectively for these nitrogen sources. This proton imbalance can be shown to be consistent with the initial assimilation steps of nitrogen from glutamate to peptide bonds which indicates that the remainder of metabolism is largely net proton balanced. These results are refined by demonstrating pH balance for growth with incrementally fed nitric acid and ammonium hydroxide. In contrast to the typical assumption of simple charge balance, each displays a slight proton uptake (around 10 % excess) that is considerably lower than urea, which displayed a molar H+ uptake per N assimilated of up to 33 %. This work illustrates details of proton imbalance that have been largely obscured in laboratory work due to use of elevated CO2 and its associated carbonate equilibrium. Combined with the recent demonstration of preferential, mutually exclusive assimilation of ammonium over nitrate in Chlorella and Chlamydomonas, these results provide the stoichiometry and dynamics of photosynthetic algae growth needed to implement large-scale pH control in the absence of buffering. 相似文献
12.
Vladislav B. Bergo Oleg A. Sineshchekov Joel M. Kralj Ranga Partha Elena N. Spudich Kenneth J. Rothschild John L. Spudich 《The Journal of biological chemistry》2009,284(5):2836-2843
Proteorhodopsins (PRs), photoactive retinylidene membrane proteins
ubiquitous in marine eubacteria, exhibit light-driven proton transport
activity similar to that of the well studied bacteriorhodopsin from halophilic
archaea. However, unlike bacteriorhodopsin, PRs have a single highly conserved
histidine located near the photoactive site of the protein. Time-resolved
Fourier transform IR difference spectroscopy combined with visible absorption
spectroscopy, isotope labeling, and electrical measurements of light-induced
charge movements reveal participation of His-75 in the proton translocation
mechanism of PR. Substitution of His-75 with Ala or Glu perturbed the
structure of the photoactive site and resulted in significantly shifted
visible absorption spectra. In contrast, His-75 substitution with a positively
charged Arg did not shift the visible absorption spectrum of PR. The mutation
to Arg also blocks the light-induced proton transfer from the Schiff base to
its counterion Asp-97 during the photocycle and the acid-induced protonation
of Asp-97 in the dark state of the protein. Isotope labeling of histidine
revealed that His-75 undergoes deprotonation during the photocycle in the
proton-pumping (high pH) form of PR, a reaction further supported by results
from H75E. Finally, all His-75 mutations greatly affect charge movements
within the PR and shift its pH dependence to acidic values. A model of the
proteorhodopsin proton transport process is proposed as follows: (i) in the
dark state His-75 is positively charged (protonated) over a wide pH range and
interacts directly with the Schiff base counterion Asp-97; and (ii)
photoisomerization-induced transfer of the Schiff base proton to the Asp-97
counterion disrupts its interaction with His-75 and triggers a histidine
deprotonation.A variety of unicellular microorganisms contain primary proton pumps that
convert solar energy into a transmembrane electrochemical proton gradient,
which is subsequently used by membrane ATP synthases to generate chemical
energy. Well known examples of such pumps are the haloarchaeal rhodopsins,
photoactive, seven-helix membrane proteins, which include the well studied
proton pump bacteriorhodopsin
(BR)4 from
Halobacterium salinarum and BR homologs in other haloarchaea.
Recently, a much larger new family of light-driven proton pumps, the
proteorhodopsins (PRs), was identified in marine proteobacteria throughout the
oceans
(1–3).
Despite the diverse properties of PRs, including different visible absorption
maxima and photocycle rates
(4–6),
they all share with BR several key conserved residues as well as an
all-trans-retinylidene chromophore in their unphotolyzed state, which
is covalently bound to transmembrane helix G via a protonated Schiff base
linkage.Many of the molecular events that occur in PRs following light activation
are similar to those of BR, including an initial ultrafast
all-trans→13-cis-retinal isomerization, which triggers
a sequence of protein conformational changes, including several intramolecular
proton transfer reactions. The two key carboxylate groups involved in proton
pumping in helix C of BR are conserved in PRs, and in the first found and most
commonly studied PR, the Monterey Bay variant eBAC31A08, also known as
green-absorbing proteorhodopsin (GPR), the helix C residues Asp-97 and Glu-108
undergo protonation changes during the photocycle similar to those of the
homologous carboxylate residues in BR. Initial FTIR studies on GPR identified
the role of Asp-97 as the Schiff base counterion and proton acceptor during
Schiff base deprotonation and concomitant M formation and Glu-108 as the
proton donor that reprotonates the Schiff base during N formation
(7,
8). Studies of other variants
indicate these roles of the two carboxylic acid residues are general in the
proteorhodopsin
family.5One major difference between BR and the PRs is the presence of a highly
conserved histidine residue at position 75, near the middle of transmembrane
helix B in the latter pigments. The His-75 homolog is not present in BR nor
thus far found in other microbial rhodopsins
(9). The proximity of His-75 to
the protein active site and specifically to the Schiff base counterion Asp-97
inferred from the x-ray crystal structure of BR suggests its involvement in
spectral tuning of the visible absorption
(10) and potentially PR
photochemical reactions. Because the pKa of histidine in
solution is close to neutral pH
(11), its imidazole group
often plays a major role in intramolecular proton transfers in enzymes,
including NADPH oxidase (12),
alcohol dehydrogenase (13),
carbonic anhydrase II (14),
and serine proteases (15).In this study we have used a combination of time-resolved FTIR difference
spectroscopy, visible absorption spectroscopy, isotope labeling, kinetic
charge displacement measurements, and site-directed mutagenesis to study the
role of His-75 in GPR. We report evidence that protonated His-75 interacts
directly with Asp-97 in the unphotolyzed protein and during the photocycle
undergoes a deprotonation in response to the protonation of Asp-97. 相似文献
13.
The functions of the four aspartic acid residues in interhelical loops at the cytoplasmic surface of bacteriorhodopsin, Asp-36, Asp-38, Asp-102, and Asp-104, were investigated by studying single and multiple aspartic acid to asparagine mutants. The same mutants were examined also with the additional D96N residue replacement. The kinetics of the M and N intermediates of the photochemical cycles of these recombinant proteins were affected only in a minor, although self-consistent, way. When residue 38 is an aspartate and anionic, it makes the internal proton exchange between the retinal Schiff base and Asp-96 about 3 times more rapid, and events associated with the reisomerization of retinal to all-trans about 3 times slower. Asp-36 has the opposite effect on these processes, but to a smaller extent. Asp-102 and Asp-104 have even less or none of these effects. Of the four aspartates, only Asp-36 could play a direct role in proton uptake at the cytoplasmic surface. In the 13 bacterioopsin sequences now available, only this surface aspartate is conserved. 相似文献
14.
The pKa value of His 57-Asp 102 couple in the active site of bovine pancreatic beta-trypsin: a molecular orbital study 总被引:1,自引:0,他引:1
The charge relay hypothesis generated a large number of theoretical and experimental studies that tested the ideas involved. Opinion based upon theoretical and experimental studies is divided on the prediction, although there are many experimental data which do not support the hypothesis. The essential feature is the proton transfer from the histidine imidazole to the aspartate. Thus, we have performed the detailed calculations of the proton transfer from His 57 to Asp 102 including the environment of the couple in protonated bovine pancreatic β-trypsin. The charge state of the His 57-Asp 102 couple is greatly influenced by the environment of the enzyme around it. In this paper, it is shown that the proton between His 57 and Asp 102 is covalently bonded to the His 57 imidazole in the protonated β-trypsin. Our MO calculations, which support the neutral-pK-histidine theory as the results, do not support the charge relay mechanism. 相似文献
15.
Photosynthetic Electron Transport Involved in PxcA-Dependent Proton Extrusion in Synechocystis sp. Strain PCC6803: Effect of pxcA Inactivation on CO2, HCO3−, and NO3− Uptake
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Masatoshi Sonoda Hirokazu Katoh Wim Vermaas George Schmetterer Teruo Ogawa 《Journal of bacteriology》1998,180(15):3799-3803
The product of pxcA (formerly known as cotA) is involved in light-induced Na+-dependent proton extrusion. In the presence of 2,5-dimethyl-p-benzoquinone, net proton extrusion by Synechocystis sp. strain PCC6803 ceased after 1 min of illumination and a postillumination influx of protons was observed, suggesting that the PxcA-dependent, light-dependent proton extrusion equilibrates with a light-independent influx of protons. A photosystem I (PS I) deletion mutant extruded a large number of protons in the light. Thus, PS II-dependent electron transfer and proton translocation are major factors in light-driven proton extrusion, presumably mediated by ATP synthesis. Inhibition of CO2 fixation by glyceraldehyde in a cytochrome c oxidase (COX) deletion mutant strongly inhibited the proton extrusion. Leakage of PS II-generated electrons to oxygen via COX appears to be required for proton extrusion when CO2 fixation is inhibited. At pH 8.0, NO3− uptake activity was very low in the pxcA mutant at low [Na+] (~100 μM). At pH 6.5, the pxcA strain did not take up CO2 or NO3− at low [Na+] and showed very low CO2 uptake activity even at 15 mM Na+. A possible role of PxcA-dependent proton exchange in charge and pH homeostasis during uptake of CO2, HCO3−, and NO3− is discussed. 相似文献
16.
《Bioscience, biotechnology, and biochemistry》2013,77(7):1364-1370
Bacteriorhodopsin (BR) is a retinal protein that functions as a light-driven proton pump. In this study, six novel mutants including K41E and D102K, were obtained to verify or rule out the possibility that residues Lys41 and Asp102 are determinants of the time order of proton release and uptake, because we found that the order was reversed in another retinal protein archaerhodopsin 4 (AR4), which had different 41th and 102th residues. Our results rule out that possibility and confirm that the pK a of the proton release complex (PRC) determines the time order. Nevertheless, mutations, especially D102K, were found to affect the kinetics of proton uptake substantially and the pK a of Asp96. Compared to the wild-type BR (BR-WT), the decay of the M intermediate and proton uptake in the photocycle was slowed about 3-fold in D102K. Hence those residues might be involved in proton uptake and delivery to the internal proton donor. 相似文献
17.
Peaks corresponding to the C(2)-protons of all four histidine residues of porcine β-trypsin were resolved in 250 MHz nuclear magnetic resonance spectra after deuteration of the slowly exchangeable N-H groups (whose resonances obscure the histidine peaks) by reversible unfolding of the protein in D2O. One of the four peaks was assigned to the charge-relay histidine in the active site of trypsin (His(57) in the bovine chymotrypsinogen numbering system). Whereas the three other histidine C(2)-peaks exhibited normal titration curves with single pK′ values of 7.20, 6.71 and 6.67, the peak assigned to His(57) had an abnormal titration curve showing two protonation steps in the pH range from 1 to 9. The first protonation with a pH′mid of 5.0 is rapid on the nuclear magnetic resonance time-scale; the second with a pH′mid of 4.5 is slow and apparently involves conformational transitions between two states having lifetimes of approximately 18 ms.In the complex between porcine β-trypsin and bovine pancreatic trypsin inhibitor (Kunitz) His(57) was found to be insensitive to pH over the range from 4 to 9 and its chemical shift resembles that of His(57) in the singly protonated charge relay of free trypsin. This result provides direct evidence that the trypsin charge relay acts as a proton acceptor in the initial catalytic step which leads to the formation of a tetrahedral complex. In the presence of equimolar bovine pancreatic trypsin inhibitor (Kunitz) the pH'mid of the conformational transition that affects the charge-relay histidine is lowered from 4.5 to approximately 3.5. 相似文献
18.
Spectral studies demonstrated that acidic pH values induce a two-step denaturation of porcine elastase, the first conformational transition occuring over the pH range 4.2–3.8, the second between pH 3.3 and 2.9. The proflavine-sensitized photooxidation of elastase in its native state, as well as in its denatured conformations, allowed us to isolate elastase derivatives selectively modified at given tryptophyl residues, hence to draw reliable conclusions about their degree of burial inside the protein matrix and their functional and conformational role. In particular, tryptophan-26 and -164 are located at the surface of the protein molecule, and their oxidation to N-formylkynurenine has no appreciable effect on the elastolytic activity and three-dimensional geometry of elastase. Tryptophan-83 is partially shielded from the aqueous environment; its modification affects only slightly the enzymic efficiency, while the tertiary structure of the protein perhaps increases its rigidity. Tryptophan-12 must be largely buried in internal regions, since its photooxidation is possible only after the native elastase structure has been extensively randomized; its indole ring appears to be of critical importance for the enzymic activity and the conformational stability of elastase. Finally, our data suggest that tryptophan-39, -132, and -232 are deeply buried; consequently, we failed to achieve the specific or preferential modification of these residues. 相似文献
19.
A bioinformatic approach was used for the identification of residues that are conserved within the Nramp family of metal transporters. Site-directed mutagenesis was then carried out to change six conserved acidic residues (i.e., Asp-34, Glu-102, Asp-109, Glu-112, Glu-154, and Asp-238) in the E. coli Nramp homolog mntH. Of these six, five of them, Asp-34, Glu-102, Asp-109, Glu-112, and Asp-238 appear to be important for function since conservative substitutions at these sites result in a substantial loss of transport function. In addition, all of the residues within the signature sequence of the Nramp family, DPGN, were also mutated in this study. Each residue was changed to several different side chains, and of ten site-directed mutations made in this motif, only P35G showed any measurable level of 54Mn2+ uptake with a Vmax value of approximately 10% of wild-type and a slightly elevated Km value. Overall, the data are consistent with a model where helix breakers in the conserved DPGN motif in TMS-1 provide a binding pocket in which Asp-34, Asn-37, Asp-109, Glu-112 (and possibly other residues) are involved in the coordination of Mn2+. Other residues such as Glu-102 and Asp238 may play a role in the release of Mn2+ to the cytoplasm or may be involved in maintaining secondary structure.This revised version was published online in June 2005 with a corrected cover date. 相似文献
20.