Intramolecular cleavage of LexA and phage lambda repressors: dependence of kinetics on repressor concentration, pH, temperature, and solvent

LexA repressor of Escherichia coli and phage lambda repressor are inactivated in vivo and in vitro by specific cleavage of an Ala-Gly peptide bond in reactions requiring RecA protein. At mildly alkaline pH, the in vitro cleavage reaction also proceeds spontaneously, suggesting that peptide bond hydrolysis is an activity of the repressors rather than of RecA. The spontaneous cleavage reaction, termed "autodigestion", has been characterized for the LexA and lambda repressors. The results show that the reaction is intramolecular. The rate of LexA autodigestion was studied over the pH range 7.15-11.77 and over the temperature range 4-46 degrees C. The logarithm of the rate constant increased linearly with pH and reached a plateau value (2.5 X 10(-3) s-1 at 37 degrees C) at pH above 10. The data closely followed a model in which a single residue side chain (apparent pK = 9.8 at 37 degrees C) must be deprotonated for the protein to show activity. Analysis of the temperature dependence gave the heat of proton dissociation as 19.9 kcal/mol and the heat of activation for hydrolysis as 15.3 kcal/mol at 25 degrees C. Autodigestion of lambda repressor, studied over the pH range 8.65-10.70 at 37 degrees C, was similar to the LexA reaction in its pH dependence, yielding a pK of 9.8. The maximum rate at 37 degrees C for lambda repressor, 6.1 X 10(-5) s-1, was 40 times slower than for LexA, a difference similar to that previously observed in vivo and in vitro for RecA-dependent cleavage reactions. There was no significant solvent deuterium isotope effect on the autodigestion of LexA. Changes in buffer composition, including high concentrations of glycine for lambda repressor and of imidazole or hydroxylamine for LexA, indicated that solvent components other than water do not participate in the rate-determining step. Removal or addition of metal ions did not significantly affect LexA autodigestion. These and other observations suggest that the deprotonated form of an amino acid side chain plays a central role in the chemistry of the cleavage reaction. The above observations establish repressor autodigestion as a member of an emerging set of biologically important self-processing reactions.     

pH Dependence of the photocycle kinetics of the E46Q mutant of photoactive yellow protein: protonation equilibrium between I1 and I2 intermediates,chromophore deprotonation by hydroxyl uptake,and protonation relaxation of the dark state

The kinetics of the photocycle of PYP and its mutants E46Q and E46A were investigated as a function of pH. E46 is the putative donor of the chromophore which becomes protonated in the I(2) intermediate. For E46Q we find that I(2) is in a pH-dependent equilibrium with its precursor I(1)' with a pK(a) of 8.15 and n = 1. From this result and from experiments with pH indicator dyes, we conclude that in the I(1)' to I(2) transition one proton is taken up from the external medium. The pK(a) of 8.15 is that of the surface-exposed chromophore in the equilibrium between I(1)' and I(2) and is close to that of the phenolate group of p-hydroxycinnamic acid. The pH-dependent I(1)'/I(2) equilibrium with associated H(+) uptake is reminiscent of the M(I)/M(II) equilibrium in the formation of the signaling state of rhodopsin. Well above this pK(a) no I(2) is formed and I(1)' returns in a pH-independent manner to the initial state P. The decay rate for the return to P via I(2) is between pH 4 and pH 8, exactly proportional to the hydroxide concentration (first order), and the deprotonation of the chromophore in this transition occurs by hydroxide uptake. Well above the pK(a) of 8.15 the apparent rate constant for the return to P is constant due to the branching from I(1)'. Complementary measurements with the pH indicator dye cresol red at pH 8.3 show that the remaining PYP molecules that still cycle via I(2) take up one proton in the formation of I(2). Together, these observations provide compelling evidence that during the photocycle the chromophore in E46Q is protonated and deprotonated from the external medium. For the yellow form of the mutant E46A the apparent rate constant for the return to P is also linear in [OH(-)] below about pH 8.3 and constant above about pH 9.5, with a pK(a) value of 8.8 for I(1)', suggesting a similar mechanism of chromophore protonation/deprotonation as in E46Q. For wild type qualitatively similar observations were made: the amplitude of I(2) decreased at alkaline pH, I(1)' and I(2) were in equilibrium, and I(1)' decayed together with the return to P. Chromophore hydrolysis prevented, however, an accurate determination of the pK(a) of I(1)'. We estimate that its value is above 11. The ground state P is in the dark in a pH-dependent equilibrium with a low-pH bleached form P(bl) with protonated chromophore. The pK(a) values for these equilibria are 4.8 and 7.9 for E46Q and E46A, respectively. When the pH is close to these pK(a)'s, the kinetics of the photocycle contains additional components in the millisecond time range. Using pH-jump stopped-flow experiments, we show that these contributions are due to the relaxation of the P/P(bl) equilibrium which is perturbed by the rapid decrease in the P concentration caused by the flash excitation of P. The condition for the occurrence of this effect is that the relaxation time of the P/P(bl) equilibrium is faster than the photocycle time.     

Effects of pH-induced variations of the charge of the transmembrane alpha-helical peptide Ac-K2(LA)12K2-amide on the organization and dynamics of the host dimyristoylphosphatidylcholine bilayer membrane

The effects of the transmembrane alpha-helical peptide Ac-K(2)(LA)(12)K(2)-amide ((LA)(12)) on the phase transition and dynamics of saturated dimyristoylphosphatidylcholine (DMPC) membranes were investigated at different pH using conventional and saturation-recovery EPR observations of phosphatidylcholine spin labels. At a peptide-to-DMPC ratio of 1/10, the main phase-transition temperature of the DMPC bilayer is decreased by 4.0 degrees C when measured at pH 7.0, by 1.6 degrees C when measured at pH 9.5, and not affected when measured at pH 11.5. This reversible pH effect is due to the subsequent neutralization of the positive charges of lysine side chains at both ends of (LA)(12). Apparent pK(a)s of the lysine side chain amino groups of (LA)(12) in DMPC bilayer are 8.6 and approximately 10.9, as compared with the pK(a) value of 10.5 for these groups when lysine is dissolved in water. Saturation-recovery curves as a function of oxygen concentration using phosphatidylcholine spin labels in DMPC bilayer containing (LA)(12) are always mono-exponential when measured at pH 7.0 and 9.5. This observation is consistent with the hypothesis that the lipid exchange rates among the bulk, boundary, and (LA)(12)-rich regions are faster than 0.5 micros, the electron spin-lattice relaxation time in the presence of molecular oxygen, suggesting that stable oligomers of (LA)(12) do not form. Neutralization of one lysine side chain positive charge on each end of the peptide significantly decreases the ordering effect of (LA)(12) on the lipid hydrocarbon chains, while its effect on the reorientational motion of terminal groups of lipid hydrocarbon chains is rather moderate. It does not affect the local diffusion-solubility product of oxygen measured in the DMPC-(LA)(12) membrane interior.     

Interhelical ion pairing in coiled coils: solution structure of a heterodimeric leucine zipper and determination of pKa values of Glu side chains

Residues of opposite charge often populate heptad positions g (heptad i on chain 1) and e' (heptad i + 1 on chain 2) in dimeric coiled coils and may stabilize the dimer by formation of interchain ion pairs. To investigate the contribution to stability of such electrostatic interactions we have designed a disulfide-linked heterodimeric zipper (AB zipper) consisting of the acidic chain Ac-E-VAQLEKE-VAQAEAE-NYQLEQE-VAQLEHE-CG-NH(2) and the basic chain Ac-E-VQALKKR-VQALKAR-NYAAKQK-VQALRHK-CG-NH(2) in which all e and g positions are occupied by either E or K/R to form a maximum of seven interhelical salt bridges. Temperature-induced denaturation experiments monitored by circular dichroism reveal a stable coiled coil conformation below 50 degrees C and in the pH range 1.2-8.0. Stability is highest at pH approximately 4.0 [DeltaG(U) (37 degrees C) = 5.18 +/- 0.51 kcal mol(-)(1)]. The solution structure of the AB zipper at pH 5.65 has been elucidated on the basis of homonuclear (1)H NMR data collected at 800 MHz [heavy atom rmsd's for the ensemble of 50 calculated structures are 0.47 +/- 0.13 A (backbone) and 0.95 +/- 0.16 A (all)]. Both chains of the AB zipper are almost entirely in alpha-helical conformation and form a superhelix with a left-handed twist. Overhauser connectivities reveal close contacts between g position residues (heptad i on chain 1) and residues d/f (heptad i on chain 1), residues a/d (heptad i + 1 on chain 1), and residue a' (heptad i + 1 on chain 2). Residues in position e (heptad i on chain 1) are in contact with residues a/b/d/f (heptad i on chain 1) and residue d' (heptad i on chain 2). These connectivities hint at a relatively defined alignment of the side chains across the helix interface. Partial H-bond formation between the functional groups of residues g and e'(+1) is observed in the calculated structures. NMR pH titration experiments disclose pK(a) values for Glu delta-carboxylate groups: 4.14 +/- 0.02 (E(1)), 4.82 +/- 0.07 (E(6)), 4.52 +/- 0.01 (E(8)), 4.37 +/- 0.03 (E(13)), 4.11 +/- 0.02 (E(15)), 4.41 +/- 0.07 (E(20)), 4.82 +/- 0.03 (E(22)), 4.65 +/- 0.04 (E(27)), 4.63 +/- 0.03 (E(29)), 4.22 +/- 0.02 (E(1)(')). By comparison with pK(a) of Glu in unfolded peptides ( approximately 4. 3 +/- 0.1), our pK(a) data suggest marginal or even unfavorable contribution of charged Glu to the stability of the AB zipper. The electrostatic energy gained from interhelical ion pairs is likely to be surpassed by hydrophobic energy terms upon protonation of Glu, due to increased hydrophobicity of uncharged Glu and, thus, better packing against apolar residues at the chain interface.     

Unraveling the catalytic mechanism of nitrile hydratases

To elucidate a detailed catalytic mechanism for nitrile hydratases (NHases), the pH and temperature dependence of the kinetic constants k(cat) and K(m) for the cobalt-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) were examined. PtNHase was found to exhibit a bell-shaped curve for plots of relative activity versus pH at pH 3.2-11 and was found to display maximal activity between pH 7.2 and 7.8. Fits of these data provided pK(E)(S1) and pK(E)(S2) values of 5.9 +/- 0.1 and 9.2 +/- 0.1 (k(cat)' = 130 +/- 1 s(-1)), respectively, and pK(E)(1) and pK(E)(2) values of 5.8 +/- 0.1 and 9.1 +/- 0.1 (k(cat)'/K(m)' = (6.5 +/- 0.1) x 10(3) s(-1) mm(-1)), respectively. Proton inventory studies indicated that two protons are transferred in the rate-limiting step of the reaction at pH 7.6. Because PtNHase is stable at 60 degrees C, an Arrhenius plot was constructed by plotting ln(k(cat)) versus 1/T, providing E(a) = 23.0 +/- 1.2 kJ/mol. The thermal stability of PtNHase also allowed DeltaH(0) ionization values to be determined, thus helping to identify the ionizing groups exhibiting the pK(E)(S1) and pK(E)(S2) values. Based on DeltaH(0)(ion) data, pK(E)(S1) is assigned to betaTyr(68), whereas pK(E)(S2) is assigned to betaArg(52), betaArg(157), or alphaSer(112) (NHases are alpha(2)beta(2)-heterotetramers). A combination of these data with those previously reported for NHases and synthetic model complexes, along with sequence comparisons of both iron- and cobalt-type NHases, allowed a novel catalytic mechanism for NHases to be proposed.     

A single protein research integrated advanced biochemistry laboratory course; spectroscopic determination of tyrosyl side chain pKa

The unique bio-analytical properties of the amino acid tyrosine (Tyr) are the focus of this experiment from the research oriented biochemistry laboratory course at our university. In the present study pK(a(1)), pK(a(2)), and pK(a(3)) values for free Tyr were estimated to be 2.30, 9.40, and 9.97, respectively, when free Tyr was titrated with 1mM NaOH and 1mM HCl using a pH meter. Spectrophotometric analysis of the phenolic side chain pK(a(3)) revealed a value of 10.14, which was consistent with the pK(a)s estimated from the pH meter. The results from this experiment will allow students to compare the free Tyr properties with those present in a protein.     

Modulation of the catalytic activity of cruzipain, the major cysteine proteinase from Trypanosoma cruzi, by temperature and pH.

Cysteine proteinases are relevant to several aspects of the parasite life cycle and of parasite-host relationships. Here, a quantitative investigation of the effect of temperature and pH on the total substrate inhibition of cruzipain, the major papain-like cysteine proteinase from Trypanosoma cruzi, is reported. Values of the apparent catalytic and inhibition parameters Km, Vmax, Vmax/Km, and K(i) for the cruzipain-catalysed hydrolysis of N-alpha-benzyloxycarbonyl-L-phenylalanyl-L-arginine-(7-amino-4-methylcoumarin) (Z-Phe-Arg-AMC) and azocasein were determined between 10.0 degrees C and 40.0 degrees C and between pH 4.5 and 8.5. Values of Km were independent of temperature and pH, whereas values of Vmax, Vmax/Km, and K(i) were temperature-dependent and pH-dependent. Over the whole pH range explored, values of logVmax, log(Vmax/Km), and logK(i) increased linearly with respect to T(-1). Values of Vmax and Vmax/Km were affected by the acid-base equilibrium of one temperature-independent ionizing group (i.e. pK(unl)' = pK(lig)' = 5.7 +/- 0.1, at 25.0 degrees C). Moreover, values of K(i) were affected by the alkaline pK shift of one ionizing group of active cruzipain (from pK(unl)" = 5.7 +/- 0.1 to pK(lig)" = 6.1 +/- 0.1, at 25.0 degrees C) upon Z-Phe-Arg-AMC binding. Values of logK(unl)', logK(lig)', and logK(lig)" were temperature-independent. Conversely, values of logK(unl)" were linearly dependent on T(-1). As a whole, total substrate inhibition of cruzipain decreased with increasing temperature and pH. These data suggest that both synthetic and protein substrates can bind to the unique active centre of cruzipain either productively or following a binding mode which results in enzyme inhibition. However, allosteric effect(s) cannot be excluded.     

Acidic-basic properties of three alanine-based peptides containing acidic and basic side chains: comparison between theory and experiment

The purpose of this work was to evaluate the effect of the nature of the ionizable end groups, and the solvent, on their acid-base properties in alanine-based peptides. Hence, the acid-base properties of three alanine-based peptides: Ac-KK-(A)(7)-KK-NH(2) (KAK), Ac-OO-(A)(7)-DD-NH(2) (OAD), Ac-KK-(A)(7)-EE-NH(2) (KAE), where A, D, E, K, and O denote alanine, aspartic acid, glutamic acid, lysine, and ornithine, respectively, were determined in water and in methanol by potentiometry. With the availability of these data, the ability of two theoretical methods to simulate pH-metric titration of those peptides was assessed: (i) the electrostatically driven Monte Carlo method with the ECEPP/3 force field and the Poisson-Boltzmann approach to compute solvation energy (EDMC/PB/pH), and (ii) the molecular dynamics method with the AMBER force field and the Generalized Born model (MD/GB/pH). For OAD and KAE, pK(a1) and pK(a2) correspond to the acidic side chains. For all three compounds in both solvents, the pK(a1) value is remarkably lower than the pK(a) of a compound modeling the respective isolated side chain, which can be explained by the influence of the electrostatic field from positively charged ornithine or lysine side chains. The experimental titration curves are reproduced well by the MD/GB/pH approach, the agreement being better if restraints derived from NMR measurements are incorporated in the conformational search. Poorer agreement is achieved by the EDMC/PB/pH method.     

Identification of the ligands to the ferric heme of Chlamydomonas chloroplast hemoglobin: evidence for ligation of tyrosine-63 (B10) to the heme

We have studied the unusual heme ligand structure of the ferric forms of a recombinant Chlamydomonas chloroplast hemoglobin and its several single-amino acid mutants by EPR, optical absorbance, and resonance Raman spectroscopy. The helical positions of glutamine-84, tyrosine-63, and lysine-87 are suggested to correspond to E7, B10, and E10, respectively, in the distal heme pocket on the basis of amino acid sequence comparison of mammalian globins. The protein undergoes a transition with a pK of 6.3 from a six-coordinate high-spin aquomet form at acidic pH to a six-coordinate low-spin form. The EPR signal of the low-spin form for the wild-type protein is absent for the Tyr63Leu mutant, suggesting that the B10 tyrosine in the wild-type protein ligates to the heme as tyrosinate. For the Tyr63Leu mutant, a new low-spin signal resembling that of alkaline cytochrome c (a His-heme-Lys species) is resolved, suggesting that the E10 lysine now coordinates to the heme. In the wild-type protein, the oxygen of the tyrosine-63 side chain is likely to share a proton with the side chain of lysine-87, suggested by the observation of a H/D sensitive resonance Raman line at 502 cm(-)(1) that is tentatively assigned as a vibrational mode of the Fe-O bond between the iron and the tyrosinate. We propose that the transition from the high-spin to the low-spin form of the protein occurs by deprotonation and ligation to the heme of the B10 tyrosine oxygen, facilitated by strong interaction with the E10 lysine side chain.     

The role of residues outside the active site: structural basis for function of C191 mutants of Escherichia coli aspartate aminotransferase

In previous kinetic studies of Escherichia coli aspartate aminotransferase, it was determined that some substitutions of conserved cysteine 191, which is located outside of the active site, altered the kinetic parameters of the enzyme (Gloss,L.M., Spencer,D. E. and Kirsch,J.F., 1996, Protein Struct. Funct. Genet., 24, 195-208). The mutations resulted in an alkaline shift of 0.6-0.8 pH units for the pK(a) of the internal aldimine between the PLP cofactor and Lys258. The change in the pK(a) affected the pH dependence of the k(cat)/K(m) (aspartate) values for the mutant enzymes. To help to understand these observations, crystal structures of five mutant forms of E.coli aspartate aminotransferase (the maleate complexes of C191S, C191F, C191Y and C191W, and C191S without maleate) were determined at about 2 A resolution in the presence of the pyridoxal phosphate cofactor. The overall three-dimensional fold of each mutant enzyme is the same as that of the wild-type protein, but there is a rotation of the mutated side chain around its C(alpha)-C(beta) bond. This side chain rotation results in a change in the pattern of hydrogen bonding connecting the mutant residue and the protonated Schiff base of the cofactor, which could account for the altered pK(a) of the Schiff base imine nitrogen that was reported previously. These results demonstrate how residues outside the active site can be important in helping determine the subtleties of the active site amino acid geometries and interactions and how mutations outside the active site can have effects on catalysis. In addition, these results help explain the surprising result previously reported that, for some mutant proteins, replacement of a buried cysteine with an aromatic side chain did not destabilize the protein fold. Instead, rotation around the C(alpha)-C(beta) bond allowed each large aromatic side chain to become buried in a nearby pocket without large changes in the enzyme's backbone geometry.     

MCCE analysis of the pKas of introduced buried acids and bases in staphylococcal nuclease

The pK(a)s of 96 acids and bases introduced into buried sites in the staphylococcal nuclease protein (SNase) were calculated using the multiconformation continuum electrostatics (MCCE) program and the results compared with experimental values. The pK(a)s are obtained by Monte Carlo sampling of coupled side chain protonation and position as a function of pH. The dependence of the results on the protein dielectric constant (ε(prot)) in the continuum electrostatics analysis and on the Lennard-Jones non-electrostatics parameters was evaluated. The pK(a)s of the introduced residues have a clear dependence on ε(prot,) whereas native ionizable residues do not. The native residues have electrostatic interactions with other residues in the protein favoring ionization, which are larger than the desolvation penalty favoring the neutral state. Increasing ε(prot) scales both terms, which for these residues leads to small changes in pK(a). The introduced residues have a larger desolvation penalty and negligible interactions with residues in the protein. For these residues, changing ε(prot) has a large influence on the calculated pK(a). An ε(prot) of 8-10 and a Lennard-Jones scaling of 0.25 is best here. The X-ray crystal structures of the mutated proteins are found to provide somewhat better results than calculations carried out on mutations made in silico. Initial relaxation of the in silico mutations by Gromacs and extensive side chain rotamer sampling within MCCE can significantly improve the match with experiment.     

Combining conformational flexibility and continuum electrostatics for calculating pK(a)s in proteins

Protein stability and function relies on residues being in their appropriate ionization states at physiological pH. In situ residue pK(a)s also provides a sensitive measure of the local protein environment. Multiconformation continuum electrostatics (MCCE) combines continuum electrostatics and molecular mechanics force fields in Monte Carlo sampling to simultaneously calculate side chain ionization and conformation. The response of protein to charges is incorporated both in the protein dielectric constant (epsilon(prot)) of four and by explicit conformational changes. The pK(a) of 166 residues in 12 proteins was determined. The root mean square error is 0.83 pH units, and >90% have errors of <1 pH units whereas only 3% have errors >2 pH units. Similar results are found with crystal and solution structures, showing that the method's explicit conformational sampling reduces sensitivity to the initial structure. The outcome also changes little with protein dielectric constant (epsilon(prot) 4-20). Multiconformation continuum electrostatics titrations show coupling of conformational flexibility and changes in ionization state. Examples are provided where ionizable side chain position (protein G), Asn orientation (lysozyme), His tautomer distribution (RNase A), and phosphate ion binding (RNase A and H) change with pH. Disallowing these motions changes the calculated pK(a).     

Structural studies of two mutants of amicyanin from Paracoccus denitrificans that stabilize the reduced state of the copper

Mutation of Pro94 to phenylalanine or alanine significantly alters the redox properties of the type I copper center of amicyanin. Each mutation increases the redox midpoint potential (E(m)) value by at least 140 mV and shifts the pK(a) for the pH dependence of the E(m) value to a more acidic value. Atomic resolution (0.99-1.1 A) structures of both the P94F and P94A amicyanin have been determined in the oxidized and reduced states. In each amicyanin mutant, an electron-withdrawing hydrogen bond to the copper-coordinating thiolate sulfur of Cys92 is introduced by movement of the amide nitrogens of Phe94 and Ala94 much closer to the thiolate sulfur than in wild-type amicyanin. This is the likely explanation for the much more positive E(m) values which result from each of these mutations. The observed decrease in the pK(a) value for the pH dependence of the E(m) value that is seen in the mutants seems to be correlated with steric hindrance to the rotation of the His95 copper ligand which results from the mutations. In wild-type amicyanin the His95 side chain undergoes a redox and pH-dependent conformational change which accounts for the pH dependence of the E(m) value of amicyanin. The reduced P94A amicyanin exhibits two alternate conformations with the positions of the copper 1.4 A apart. In one of these conformations, a water molecule appears to have replaced Met98 as a copper ligand. The relevance of these structures to the electron transfer properties of P94F and P94A amicyanin are also discussed.     

High apparent dielectric constant inside a protein reflects structural reorganization coupled to the ionization of an internal Asp

The dielectric properties of proteins are poorly understood and difficult to describe quantitatively. This limits the accuracy of methods for structure-based calculation of electrostatic energies and pK(a) values. The pK(a) values of many internal groups report apparent protein dielectric constants of 10 or higher. These values are substantially higher than the dielectric constants of 2-4 measured experimentally with dry proteins. The structural origins of these high apparent dielectric constants are not well understood. Here we report on structural and equilibrium thermodynamic studies of the effects of pH on the V66D variant of staphylococcal nuclease. In a crystal structure of this protein the neutral side chain of Asp-66 is buried in the hydrophobic core of the protein and hydrated by internal water molecules. Asp-66 titrates with a pK(a) value near 9. A decrease in the far UV-CD signal was observed, concomitant with ionization of this aspartic acid, and consistent with the loss of 1.5 turns of alpha-helix. These data suggest that the protein dielectric constant needed to reproduce the pK(a) value of Asp-66 with continuum electrostatics calculations is high because the dielectric constant has to capture, implicitly, the energetic consequences of the structural reorganization that are not treated explicitly in continuum calculations with static structures.     

Multi-modality of pI distribution in whole proteome

Multi-modality of pI distribution is a common feature in different whole proteomes. Some researchers considered it relate to the proteins with different subcellular locations, indicating the result of natural selection. We explored the pI distribution of predicted proteomes (including animals, plants, bacterium, archaeans) and random proteome [random protein sequences constructed according to the special amino acid composition and molecular weight (MW) distribution of human predicted proteome]. Our results suggest that the multi-modality is the result of discrete pK(R) values for different amino acids. Amino acid composition and MW distribution of a proteome also contributes to the specific pI distribution. Although protein subcellular location was related to pI value, our analyses revealed that comparing with the random proteome, neither the multi-modality phenomenon nor the distribution bias of pI values is caused by subcellular location. It seems that the multi-modality distribution is just a mathematical fun. The blank region near the neutral pI was caused by the absence of amino acids with neutral pK(R), and suggests that the selection of amino acids with ionizable side chain might be restricted by the requirement for a special pH environment during the origin of life. From this point of view, the special distribution was the result of natural selection.     

The CXC motif: a functional mimic of protein disulfide isomerase

Protein disulfide isomerase (PDI) utilizes the active site sequence Cys-Gly-His-Cys (CGHC; E degrees ' = -180 mV) to effect thiol-disulfide interchange during oxidative protein folding. Here, the Cys-Gly-Cys-NH(2) (CGC) peptide is shown to have a disulfide reduction potential (E degrees ' = -167 mV) that is close to that of PDI. This peptide has a thiol acid dissociation constant (pK(a) = 8.7) that is lower than that of glutathione. These attributes endow the CGC peptide with substantial disulfide isomerization activity. Escherichia coli thioredoxin (Trx) utilizes the active site sequence Cys-Gly-Pro-Cys (CGPC; E degrees ' = -270 mV) to effect disulfide reduction. Removal of the proline residue from the Trx active site yields a CGC active site with a greatly destabilized disulfide bond (E degrees ' >or= -200 mV). The DeltaP34 variant retains high conformational stability and remains a substrate for thioredoxin reductase. In contrast to the reduced form of the wild-type enzyme, the reduced form of DeltaP34 Trx has disulfide isomerization activity, which is 25-fold greater than that of the CGC peptide. Thus, the rational deletion of an active site residue can bestow a new and desirable function upon an enzyme. Moreover, a CXC motif, in both a peptide and a protein, provides functional mimicry of PDI.     

Isolation and physico-chemical characterization of a cytochrome c from the methylotrophic yeast Hansenula polymorpha

Cytochrome c from the methylotrophic yeast Hansenula polymorpha was isolated and purified to homogeneity for the first time. The final yield of the highly purified protein from 1.4 kg (wet weight) cells was about 20 mg. The hemoprotein has an apparent molecular mass of 12 kDa and isoelectric point (pI) of 9.3. The purified protein was characterized by electronic, EPR and NMR spectroscopies. The redox potential of the cytochrome, E degrees, measured by cyclic voltammetry measurements at neutral pH, is 0.302 V. Both NMR spectroscopy and electrochemical measurements confirm the presence in the solution of several acid-base equilibria, the most pronounced being characterized by a pK(a) of 8.3. The latter pK(a) was attributed to the detachment of the iron(III) ion-coordinated methionine and its replacement by a lysine residue. The electrochemically derived thermodynamic parameters for neutral and alkaline protein species (DeltaS degrees (rc) and DeltaH degrees (rc)) were obtained from the temperature dependence of the redox potential.     

Investigation of the active site of papain with fluorescent probes

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD chloride) and 7-(2'-hydroxyethylthio)-NBD (obtained from NBD chloride and mercaptoethanol) undergo a reversible spectral change in alkaline solution that depends respectively on a single apparent pK(a) 9.76 (at 25 degrees C) and 8.81 (at 32 degrees C). In acid solution however no spectral change was observed. NBD chloride reacts slowly with papain at pH7, but the rate of inhibition increases at lower pH and depends on an apparent pK(a) of 3.7 (at 35 degrees C), which has been tentatively assigned to the carboxyl group of aspartic acid-158. The spectral properties of NBD-papain indicate that the thiol group of cysteine-25 is the site of reaction. The intensity of the fluorescence-emission spectrum of NBD-papain depends on a single pK(a) of 4.2 (at 26.7 degrees C). The intensity of the fluorescence-emission spectrum of the mixed disulphide formed from papain and 7-(2'-mercaptoethylamino)-NBD (obtained from NBD chloride and cysteamine) depended on a single pK(a) of 3.94 in water and 3.89 in aq. 19.2% (v/v) dioxan (at 27 degrees C). This small change to lower pK(a) value in a medium of lower dielectric constant is characteristic of a cationic acid. The only acid of this type in the active-site region is the conjugate acid of histidine-159.     

pH dependence of the binding constants of N-acetylglucosamine monomers to hen and turkey egg-white lysozymes.

The binding constants of N-acetylglucosamine (G1cNAc) and its methyl alpha- and beta- glycosides to hen and turkey egg-white lysozymes [EC 3.2.1.17], in the latter of which Asp 101 is replaced by Gly, were determined at various pH values by measuring changes in the circular dichroic (DC) band at 295 nm. The binding of beta-methyl-G1cNAc to turkey and hen lysozymes perturbed the pK value of Glu 35 from 6.0 to 6.5, the pK value of Asp 52 from 3.5 to 3.9, and the pK value of Asp 66 from 1.3 to 0.7. In addition, perturbation of the pK value of Asp 101 from 4.4 to 4.0 was observed in the binding of this saccharide to hen lysozyme. The binding of alpha-methyl-GlcNAc to hen and turkey lysozymes perturbed the pK value of Glu 35 to the alkaline side by about 0.5 pH unit, the pK value of Asp 66 to the acidic side by about 0.5 pH unit, and the pK value (4.4) of an ionizable group to the acidic side by about 0.6 pH unit. The last ionizable group was tentatively assigned to Asp 48. The pK value of Asp 52 was not perturbed by the binding of this saccharide. The pH dependence curves for the binding of GlcNAc to hen and turkey lysozymes were very similar and it was suggested that Asp 48, in addition to Asp 66, Asp 52, and Glu 35, is perturbed by the binding of GlcNAc.     

Structural features of luliberin (luteinising hormone-releasing factor) inferred from fluorescence measurements.

The fluorescence and excitation spectra of luliberin (luteinizing hormone-releasing factor) in 0.005 M aqueous ammonium acetate are identical in shape to those of N-acetyltryptophan amide and are related to the indole side chain of Trp3. The change of fluoresecence intensity of luliberin with pH was measured in the range of pH 4-11. The increase of pH from 4 to 7.5 is followed by about 50% increase in fluorescence intensity due to deprotonation of the imidazolium side chain of His2. The fluorimetric titration curve in this pH region reveals a pK value for His2 of 5.95. Increasing of pH from 8 to 11 results in about 40% quenching of the fluorescence due to electronic energy transfer from the excited indole of Trp3 to the phenolate side chain of Tyr5. The pK value of Tyr5, obtained independently from the fluorimetric and photometric titrations indicate that at pH 7-8 luliberin contains only one charged residue, Arg8, which is in close vicinity to both His2 and Tyr5. The side chains of His2, Tyr5 and Arg8 presumably form a combined unit which may play an active role in the hormone action. Trp3 is at a maximal distance from this unit and may thus act as an independent active unit.