Effect of low pH on the conformation of Pseudomonas exotoxin A

Previously we examined factors involved in the entry mechanism of Pseudomonas exotoxin A (PTx) at the level of lipid-protein interactions (Farahbakhsh, Z. T., Baldwin, R. L., and Wisnieski, B. J. (1986) J. Biol. Chem. 261, 11404-11408). Exposure to a low pH environment appears to be an obligatory trigger of the entry pathway. In this report we describe the effect of pH upon the conformation of PTx. We have found that the intrinsic fluorescence of PTx is strongly dependent on pH, decreasing between pH 7.4 and 4.0 with a red shift in the emission lambda max. The changes are reversible and associated with the acquisition of a binding site for the fluorescent dye 1-anilino-8-naphthalenesulfonic acid (ANS). The fluorescence intensity of ANS in the presence of PTx increases with decreasing pH and is accompanied by a blue shift in emission spectra, indicative of exposure of hydrophobic surfaces. These changes are also reversible. Both the intrinsic fluorescence and ANS binding profiles show a dramatic dependence on pH, with the transitions centered on pH 5.0 and 4.5, respectively. Circular dichroism studies reveal a 9% decrease in alpha-helicity between pH 7.7 and 4. The susceptibility of toxin to trypsin cleavage is also a function of pH, increasing with decreasing pH. The pH 7.4 cleavage profile is regained when the acid-treated samples are brought back to pH 7.4. The conformational changes observed in these pH shift experiments are likely to be physiologically significant because the conditions closely resemble those that the toxin would encounter if entry into the cytoplasm of a cell involves escape from an endosomal compartment.     

Human erythrocyte glutathione reductase: pH dependence of kinetic parameters

Human erythrocyte glutathione reductase catalyzes the pyridine nucleotide dependent reduction of oxidized glutathione (GSSG). The pH dependence of the kinetic parameters V and V/K for three reduced pyridine nucleotide substrates, the Ki's for three competitive inhibitors (versus NADPH), and the temperature dependence of the V pH profile have been determined. Below pH 8, V and V/K for NADPH, 2',3'-cyclic-NADPH, and NADH are pH independent. In the basic pH region, both V and V/K for the three substrates are pH dependent. All three of the V profiles decrease with increasing pH as a group with a pKa of approximately 9.2 is titrated. The V/K profiles for NADPH, 2',3'-cyclic-NADPH, and NADH decrease at high pH as a group with a pKa of greater than 9.8, 8.9, and 8.8, respectively, is deprotonated. The Ki's for ATP-ribose and 2',5'-ADP are pH independent below pH 8 but increase in the basic region as a group with a pKa of about 8.8 and 8.5, respectively, is deprotonated. The Ki of AADP is pH independent between pH 6 and 9. These studies suggest that binding interactions between the 2'-phosphate of NADPH and the enzyme are predominately nonionic. The temperature dependence of the pK observed in all V pH profiles allows the calculation of an enthalpy of ionization of 3.2 kcal/mol for this group. The high pK and low enthalpy of ionization suggest that the protonation state of the His-467'-Glu-472' ion pair observed in the structure of human erythrocyte glutathione reductase influences proton-transfer steps occurring in the oxidative half-reaction.     

Regulation of the formation of acid phosphatases by inorganic phosphate in Aspergillus ficuum

Two types of extracellular acid phosphatases are synthesized by Aspergillus ficuum NRRL 3135: a nonspecific orthophosphoric monoester phosphohydrolase (EC 3.1.3.2) with an optimum pH of 2.0, and an enzyme with restricted specificity, a mesoinositol-hexaphosphate phosphohydrolase (EC 3.1.3.8; phytase) with an optimum pH of 5.5. Although the pH 5.5 enzyme is termed a phytase, both enzymes hydrolyze phytin. Synthesis of the enzymes is repressed by high orthophosphate concentrations in the fermentation medium. The highest total level for each enzyme is synthesized in low orthophosphate medium. In high orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme. In low orthophosphate medium, more pH 5.5 enzyme is produced than pH 2.0 enzyme during the early stages of growth, but the reverse occurs after 5 days. The enzymes are differentiated by heat denaturation at acid and alkaline pH levels. They are separated into two distinct fractions on Sephadex G-100 followed by carboxymethylcellulose column chromatography. This indicates that the two enzymes are structurally different. The K(m) for both enzymes is 1.25 mm when calcium phytate is the substrate. Orthophosphate competitively inhibits the pH 2.0 (K(i) = 1.1 x 10(-2)m) but not the pH 5.5 phosphatase. Neither enzyme is denatured by 50% (w/v) urea or inhibited by 0.01 m tartrate. Thus, they differ from human prostatic phosphatase.     

Characterization of the transition-state structure of the reaction of kanamycin nucleotidyltransferase by heavy-atom kinetic isotope effects

The transition-state structure for the reaction catalyzed by kanamycin nucleotidyltransferase has been determined from kinetic isotope effects. The primary (18)O isotope effects at pH 5.7 (close to the optimum pH) and at pH 7.7 (away from the optimum pH) are respectively 1.016 +/- 0.003 and 1.014 +/- 0.002. Secondary (18)O isotope effects of 1.0033 +/- 0.0004 and 1.0024 +/- 0.0002 for both nonbridge oxygen atoms were measured respectively at pH 5.7 and 7.7. These isotope effects are consistent with a concerted reaction with a slightly associative transition-state structure.     

Sites of direct and indirect halogenation of albumin.

The sites of radiohalogenation in proteins vary with the labeling method and the pH of the labeling reaciton. We have directly halogenated albumin with carrier-free radioiodide by three methods (pH range 2.2--9.3), and with carrier-free radiobromide by the chloroperoxidase method (pH range 2.2--4.6). Albumin was also indirectly halogenated by attaching a radioiodinated acylating agent, N-succinimidyl-3-(4-hydroxyphenyl) propionate (SHPP). The labeled proteins were proteolyzed enzymatically at neutral pH and the labeled amino acids produced were analyzed by liquid chromatography. Iodination at pH 7 yielded predominantly monoiodotyrosine, but at lower pH, fewer tyrosyl residues are labeled and a greater number of unstable sulfur-iodine bonds are formed at cysteinyl residues. Bromination with chloroperoxidase resulted in a high degree of labeling of cysteinyl residues at pH 2.8, the condition for optimum activity of this halogenating enzyme. Indirect halogenation with SHPP resulted in labeling of mid-chain lysyl, histidyl and tyrosyl residues.     

pH modification of human T-type calcium channel gating

External pH (pH(o)) modifies T-type calcium channel gating and permeation properties. The mechanisms of T-type channel modulation by pH remain unclear because native currents are small and are contaminated with L-type calcium currents. Heterologous expression of the human cloned T-type channel, alpha1H, enables us to determine the effect of changing pH on isolated T-type calcium currents. External acidification from pH(o) 8.2 to pH(o) 5.5 shifts the midpoint potential (V(1/2)) for steady-state inactivation by 11 mV, shifts the V(1/2) for maximal activation by 40 mV, and reduces the voltage dependence of channel activation. The alpha1H reversal potential (E(rev)) shifts from +49 mV at pH(o) 8.2 to +36 mV at pH(o) 5.5. The maximal macroscopic conductance (G(max)) of alpha1H increases at pH(o) 5.5 compared to pH(o) 8.2. The E(rev) and G(max) data taken together suggest that external protons decrease calcium/monovalent ion relative permeability. In response to a sustained depolarization alpha1H currents inactivate with a single exponential function. The macroscopic inactivation time constant is a steep function of voltage for potentials < -30 mV at pH(o) 8.2. At pH(o) 5.5 the voltage dependence of tau(inact) shifts more depolarized, and is also a more gradual function of voltage. The macroscopic deactivation time constant (tau(deact)) is a function of voltage at the potentials tested. At pH(o) 5.5 the voltage dependence of tau(deact) is simply transposed by approximately 40 mV, without a concomitant change in the voltage dependence. Similarly, the delay in recovery from inactivation at V(rec) of -80 mV in pH(o) 5.5 is similar to that with a V(rec) of -120 mV at pH(o) 8.2. We conclude that alpha1H is uniquely modified by pH(o) compared to other calcium channels. Protons do not block alpha1H current. Rather, a proton-induced change in activation gating accounts for most of the change in current magnitude with acidification.     

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantitative relationships of invertebrates to pH in Norwegian river systems

The invertebrate fauna has been surveyed for twenty one unlimed generally acidic river systems in Norway. The data consist of 180 samples and 127 invertebrate taxa and associated water chemistry data (pH, calcium, acid neutralizing capacity, total aluminium, and conductivity). Multivariate numerical methods are used to quantify the relationships between aquatic invertebrates and water chemistry. Detrended canonical correspondence analysis (DCCA) shows one dominant axis of variation with high correlations for pH and aluminium. DCCA axis 2 is significantly correlated with calcium. The predictive abilities of invertebrates to pH are explored by means of weighted averaging (WA) regression and calibration and weighted averaging partial-least-squares regression (WA-PLS). The performance of the methods is reported in terms of the root mean square error of prediction (RMSEP) of (observed pH-inferred pH). Bootstrapping and leave-one-out jackknifing are used as cross-validation procedures. The predictive abilities of invertebrates are good (RMSEP_boot for WA = 0.309 pH units). Comparison of the invertebrates with diatom studies shows that invertebrates are as good predictors of modern pH as diatoms are. RMSEP_jack shows that WA-PLS improves the predictive abilities. Indicator taxa for pH are found by Gaussian regression. Anisoptera, Agrypnia obsoleta, Leptophlebia marginata, Sialis lutaria, and Zygoptera have significant sigmoidal curves where abundances increase with decreasing pH. Cyrnus flavidus shows a significant unimodal response and has an estimated optimum in the acid part of the gradient. Isoperla spp. and Ostracoda show significant sigmoidal responses where abundances increase with increasing pH. Amphinemura borealis, Diura nanseni, Isoperla grammatica, I. obscura, and Siphonoperla burmeisteri show significant unimodal responses and have high pH optima. Many taxa do not have statistically significant unimodal or sigmoidal curves, but are found by WA to be characteristic of either high pH or low pH. These results suggest that a combined use of Gaussian regression and direct gradient analysis is needed to get a full overview of potential indicator taxa.     

Inferring pH from diatoms: a comparison of old and new calibration methods

Two new methods for inferring pH from diatoms are presented. Both are based on the observation that the relationships between diatom taxa and pH are often unimodal. The first method is maximum likelihood calibration based on Gaussian logit response curves of taxa against pH. The second is weighted averaging. In a lake with a particular pH, taxa with an optimum close to the lake pH will be most abundant, so an intuitively reasonable estimate of the lake pH is to take a weighted average of the pH optima of the species present.Optima and tolerances of diatom taxa were estimated from contemporary pH and proportional diatom counts in littoral zone samples from 97 pristine soft water lakes and pools in Western Europe. The optima showed a strong relation with Hustedt's pH preference groups. The two new methods were then compared with existing calibration methods on the basis of differences between inferred and observed pH in a test set of 62 additional samples taken between 1918 and 1983. The methods were ranked in order of performance as follows (between brackets the standard error of inferred pH in pH units); maximum likelihood (0.63) > weighted averaging (0.71) = multiple regression using pH groups (0.71) = the Gasse & Tekaia method (0.71) > Renberg & Hellberg's Index B (0.83) » multiple regression using taxa (2.2). The standard errors are larger than those usually obtained from surface sediment samples. The relatively large standard may be due to seasonal variation and to the effects of other factors such as humus content. The maximum likelihood method is statistically rigorous and can in principle be extended to allow for additional environmental factors. It is computer intensive however. The weighted averaging approach is a good approximation to the maximum likelihood method and is recommended as a practical and robust alternative.     

Resonance Raman evidence for the mechanism of the allosteric control of O2-binding in a cobalt-substituted monomeric insect hemoglobin.

The substitution of iron for cobalt in the monomeric insect hemoglobin CTT (Chironomus thummi thummi) III does not alter the Bohr effect for O2-binding. The cobalt substitution in this hemoglobin allows us to identify not only the O-O and Co-O2 stretching mode but also the Co-O-O bending mode by resonance Raman spectroscopy. The assignments were made via 16O2/18O2 isotope exchange. The modes associated with the Co-O-O moiety are pH-dependent. These pH-induced changes of the resonance Raman spectra are correlated with the t = r conformation transition. At high pH (high-affinity state) two unperturbed O-O stretching modes are observed at 1,068 cm-1 (major component) and 1,093 cm-1 (minor component) for the 18O2 complex. These frequencies correspond to split modes at 1,107 cm-1 and 1,136 cm-1 and an unperturbed mode at approximately 1,153 cm-1 for the 16O2 complex. At low pH (low-affinity state) the minor component becomes the major component and vice versa. The Co-O2 stretching frequency varies for approximately 520 cm-1 (pH 5.5) to 537 cm-1 (pH 9.5) indicating a stronger (hence shorter) Co-O2 bond in the high-affinity state. On the other hand, the O-O bond is weakened upon the conversion of the low- to the high-affinity state. The Co-O-O bending mode changes from 390 cm-1 (pH 9.5) to 374 cm-1 (pH 5.5). In the deoxy form the resonance Raman spectra are essentially pH-insensitive except for a vinyl mode at 414 cm-1 (pH 5.5), which is shifted to 416 cm-1 (pH 5.5).     

Studies of the pH dependence of the formation of binary and ternary complexes with liver alcohol dehydrogenase

The association of the coenzyme NAD+ to liver alcohol dehydrogenase (LADH) is known to be pH dependent, with the binding being linked to the shift in the pK of some group on the protein from a value of 9-10, in the free enzyme, to 7.5-8 in the LADH-NAD+ binary complex. We have further characterized the nature of this linkage between NAD+ binding and proton dissociation by studying the pH dependence (pH range 6-10) of the proton release, delta n, and enthalpy change, delta Ho(app), for formation of both binary (LADH-NAD+) and ternary (LADH-NAD+-I, where I is pyrazole or trifluoroethanol) complexes. The pH dependence of both delta n and delta Ho(app) is found to be consistent with linkage to a single acid dissociating group, whose pK is perturbed from 9.5 to 8.0 upon NAD+ binding and is further perturbed to approximately 6.0 upon ternary complex formation. The apparent enthalpy change for NAD+ binding is endothermic between pH 7 and pH 10, with a maximum at pH 8.5-9.0. The pH dependence of the delta Ho(app) for both binary and ternary complex formation is consistent with a heat of protonation of -7.5 kcal/mol for the coupled acid dissociating group. The intrinsic enthalpy changes for NAD+ binding and NAD+ plus pyrazole binding to LADH are determined to be approximately 0 and -11.0 kcal/mol, respectively. Enthalpy change data are also presented for the binding of the NAD+ analogues adenosine 5'-diphosphoribose and 3-acetylpyridine adenine dinucleotide.     

Kinetic properties of pulmonary angiotensin-converting enzyme. Hydrolysis of hippurylglycylglycine.

Some of the kinetic properties of angiotensin-converting enzyme (peptidyl-dipeptide hydrolase, EC 3.4.15.1) purified from hog lung have been determined using hippurylglycylglycine as substrate. The effects of pH and ionic environment on enzyme activity are complex and interdependent. At 0.1 M NaCl, the pH-activity curve shows an abrupt decrease in V/Km as the pH rises from 6 to 6.5, implying that ionization of a group in the enzyme with a pK in this range aids in binding of the substrate. Chloride is required for enzyme activity; there are two phases in the effect of NaCl. At both pH 6 AND 8, THE FIRST PHASE (UP TO 0.1 M NaCl) is activation. The second phase (above 0.1 M) at pH 6 is inhibition, while at pH 8 there is further activation which appears to be dependent upon ionic strength rather than a specific Cl-effect. Activation by cobalt and inhibition by EDTA are somewhat more effective at pH 6 than at pH 8. The nonapeptide inhibitor less than Glu-Trp-Pro-Arg-Pro-Gln-Ile-Pro-Pro is nearly equipotent at both pH 6 and 8, but Arg-Pro-Pro is more inhibitory at pH 8 than at pH 6.     

Electron transfer to nitrogenase. Characterization of flavodoxin from Azotobacter chroococcum and comparison of its redox potentials with those of flavodoxins from Azotobacter vinelandii and Klebsiella pneumoniae (nifF-gene product).

Flavodoxin in the hydroquinone state acts as an electron donor to nitrogenase in several nitrogen-fixing organisms. The mid-point potentials for the oxidized-semiquinone and semiquinone-hydroquinone couples of flavodoxins isolated from facultative anaerobe Klebsiella pneumoniae (nifF-gene product, KpFld) and the obligate aerobe Azotobacter chroococcum (AcFld) were determined as a function of pH. The mid-point potentials of the semiquinone-hydroquinone couples of KpFld and AcFld are essentially independent of pH over the range pH 7-9, being -422 mV and -522 mV (normal hydrogen electrode) at pH 7.5 respectively. The mid-point potentials of the quinone-semiquinone couples at pH 7.5 are -200 mV (KpFld) and -133 mV (AcFld) with delta Em/pH of -65 +/- 4 mV (KpFld) and -55 +/- 2 mV (AcFld) over the range pH 7.0-9.5. This indicates that reduction of the quinone is coupled to protonation to yield a neutral semiquinone. The significance of these values with respect to electron transport to nitrogenase is discussed. The amino acid compositions, the N- and C-terminal amino acid sequences and the u.v.-visible spectra of KpFld and AcFld were determined and are compared with published data for flavodoxins isolated from Azotobacter vinelandii.     

Physico-chemical studies of a DNA triplex containing a new ferrocenemethyl-thymidine residue in the third strand

The stability of a 16-mer DNA triple helix containing a 3-N(ferrocenemethyl)-thymidine residue in the third strand has been investigated in comparison with the unmodified triplex of the same sequence. A complete physico-chemical characterization of the two triple helices on changing the pH by means of calorimetry, circular dichroism and molecular modeling is therefore reported. The thermodynamic parameters were obtained in the pH range 5.5-7.2 by differential scanning calorimetry (DSC). For both triplexes the T(m) and Delta H degrees (T(m)) values increase on decreasing the pH. In the pH range 7.2-6.0 the triplex containing the ferrocenemethyl nucleoside is less stable than the unmodified one, whereas the modified triplex becomes more stable at pH 5.5. Such difference in stability at each pH value is overwhelmingly enthalpic in origin. CD spectra show conformational changes on decreasing the pH for both the triplexes. By spectroscopic pH titration the apparent pK(a) values of the cytosines in the two triplexes could be estimated, with the cytosines in the TFO containing the ferrocenemethyl residue having lower apparent pK(a) values. These results are consistent with the calorimetric data, showing a decrease of the thermodynamic parameters in the pH range 7.2-6.0 and an increase at pH 5.5 for the ferrocenylated triplex with respect to the unmodified one. The thermodynamic and spectroscopic data are also discussed in relation to molecular models.     

Redox potentials of flavocytochromes c from the phototrophic bacteria, Chromatium vinosum and Chlorobium thiosulfatophilum.

The redox potentials of flavocytochromes c (FC) from Chromatium vinosum and Chlorobium thiosulfatophilum have been studied as a function of pH. Chlorobium FC has a single heme which has a redox potential of +98 mV at pH 7 (N = 1) that is independent of pH between 6 and 8. The average two-electron redox potential of the flavin extrapolated to pH 7 is +28 mV and decreases 35 mV/pH between pH 6 and 7. The anionic form of the flavin semiquinone is stabilized above pH 6. The redox potential of Chromatium FC is markedly lower than for Chlorobium. The two hemes in Chromatium FC appear to have a redox potential of 15 mV at pH 7 (N = 1), although they reside in very different structural environments. The hemes of Chromatium FC have a pH-dependent redox potential, which can be fit in the simplest case by a single ionization with pK = 7.05. The flavin in Chromatium FC has an average two-electron redox potential of -26 mV at pH 7 and decreases 30 mV/pH between pH 6 and 8. As with Chlorobium, the anionic form of the flavin semiquinone of Chromatium FC is stabilized above pH 6. The unusually high redox potential of the flavin, a stabilized anion radical, and sulfite binding to the flavin in both Chlorobium and Chromatium FCs are characteristics shared by the flavoprotein oxidases. By analogy with glycolate oxidase and lactate dehydrogenase for which there are three-dimensional structures, the properties of the FCs are likely to be due to a positively charged amino acid side chain in the vicinity of the N1 nitrogen of the flavin.     

A Raman spectroscopic study of the interaction of divalent metal ions with adenine moiety of adenosine 5'-triphosphate.

Raman spectra of ATP at various pH values are affected by addition of equimolar solution of divalent metal ions such as Ca2+, Mg2+, Co2+, Cu2+, and Hg2+. The changes in frequency and intensity have been used to construct models describing the nature of metal-adenine and metal-triphosphate interactions under different conditions. The metal ions are found to co-ordinate the triphosphate group in the entire pH range studies (pH to 12). Calcium (II) and magnesium (II) interact strongly with the phosphate moiety at neutral pH, although a weak interaction with the ring occur at low pH values. Around neutrality, several Raman spectral changes are observed to implicate the interaction of cobalt (II) ion with the five-membered ring of the adenine. The changes in Raman frequency are too small to suggest a direct Co(II)-N7 binding. At least six different Cu(II)-ATP species are identified between pH 3 and 12. At pH approximately 7.0 Raman data are explained better by Cu(II) interacting with N7 simultaneously with the amino group of the adenine ring. However, a Cu(II) binding to N3 at pH 10 to 11 is indicated by the enhancement of the 760 and 1360 cm-1 vibrations. At neutral pH, mercury (II) ion shows a direct coordination at N1 while at low pH with N1 blocked by protonation, mercury (II) does not interact with the adenine moiety.     

The pH dependence of pre-steady-state and steady-state kinetics for the papain-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester

Pre-steady-state and steady-state kinetics of the papain (EC 3.4.22.2)-catalyzed hydrolysis of N-alpha-carbobenzoxyglycine p-nitrophenyl ester (ZGlyONp) have been determined between pH 3.0 and 9.5 (I = 0.1 M) at 21 +/- 0.5 degrees C. The results are consistent with the minimum three-step mechanism involving the acyl X enzyme intermediate E X P: (Formula: see text). The formation of the E X S complex may be regarded as a rapid pseudoequilibrium process; the minimum values for k+1 are 8.0 microM-1 s-1 (pH less than or equal to 3.5) and 0.40 microM-1 s-1 (pH greater than 6.0), and that for k-1 is 600 s-1 (pH independent). The pH profile of k+2/Ks (= kcat/Km; Ks = k-1/k+1) reflects the ionization of two groups with pK' values of 4.5 +/- 0.1 and 8.80 +/- 0.15 in the free enzyme. The pH dependence of k+2 and k+3 (measured only at pH values below neutrality) implicates one ionizing group in the acylation and deacylation step with pK' values of 5.80 +/- 0.15 and 3.10 +/- 0.15, respectively. As expected from the pH dependences of k+2/Ks (= kcat/Km) and k+2, the value of Ks changes with pH from 7.5 X 10(1) microM (pH less than or equal to 3.5) to 1.5 X 10(3) microM (pH greater than 6.0). Values of k-2 and k-3 are close to zero over the whole pH range explored (3.0 to 9.5). The pH dependence of kinetic parameters indicates that at acid pH values (less than or equal to 3.5), the k+2 step is rate limiting in catalysis, whereas for pH values higher than 3.5, k+3 becomes rate limiting. The observed ionizations probably reflect the acid-base equilibria of residues involved in the catalytic diad of papain, His159-Cys25. Comparison with catalytic properties of ficins and bromelains suggests that the results reported here may be of general significance for cysteine proteinase catalyzed reactions.     

Modeling the effects of sodium chloride, acetic acid, and intracellular pH on survival of Escherichia coli O157:H7

Microbiological safety has been a critical issue for acid and acidified foods since it became clear that acid-tolerant pathogens such as Escherichia coli O157:H7 can survive (even though they are unable to grow) in a pH range of 3 to 4, which is typical for these classes of food products. The primary antimicrobial compounds in these products are acetic acid and NaCl, which can alter the intracellular physiology of E. coli O157:H7, leading to cell death. For combinations of acetic acid and NaCl at pH 3.2 (a pH value typical for non-heat-processed acidified vegetables), survival curves were described by using a Weibull model. The data revealed a protective effect of NaCl concentration on cell survival for selected acetic acid concentrations. The intracellular pH of an E. coli O157:H7 strain exposed to acetic acid concentrations of up to 40 mM and NaCl concentrations between 2 and 4% was determined. A reduction in the intracellular pH was observed for increasing acetic acid concentrations with an external pH of 3.2. Comparing intracellular pH with Weibull model predictions showed that decreases in intracellular pH were significantly correlated with the corresponding times required to achieve a 5-log reduction in the number of bacteria.     

Resonance Raman spectroscopic studies of adriamycin and copper(II)-adriamycin and copper(II)-adriamycin-DNA complexes

Characteristic resonance Raman spectra are observed on ionization of the phenolic groups in adriamycin. On the basis of these results, vibrational assignments for the Raman bands of adriamycin are reported. Distinct Raman spectra are observed for Cu(II)-adriamycin complexes at pH approximately 5 and pH approximately 13. The data indicate that at lower pH a bis complex of Cu(II) is formed, which transforms to a polymeric Cu(II) chelate at higher pH. Upon interaction of the metal-drug complex with calf thymus DNA at pH approximately 5, a ternary complex is formed in which the Cu(II)-complexed adriamycin is intercalated into DNA.     

Anomalous pH dependence of the reactions of carbenicillin and sulbenicillin with Bacillus cereus beta-lactamase I. Influence of the alpha-substituent charge on the kinetic parameters

The pH dependence of k(cat) for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of carbenicillin(VI), which differs from benzylpenicillin (I) in having a carboxylic moiety alpha to the phenyl ring, exhibits a profile consistent with a model in which the alpha-COOH and alpha-COO forms of the ES complex turn over with respective rate constants of 2152 s(-1) and 384 s(-1). The pK(a)(app) for the alpha-COOH is shifted from 3.2 in solution to 6.1 in the ES complex. The normalized k(cat)/K(m) vs. pH profile for VI is not superimposable on that of I, indicating that both the neutral and anionic forms of the carboxyl moiety of VI combine with the enzyme to give the first irreversibly formed complex, presumably the acyl-enzyme. Quantitative accord with the kinetic data is achieved only through fitting to a model where kinetically significant proton transfer in the ES complex is permitted. The second-order rate constants for the reaction of the enzyme with the alpha-COOH and alpha-COO forms of VI are 2.2 x 10(8) M(-1) s(-1) and 3.8 x 10(6) M(-1) s(-1), respectively. The high value for the alpha-COOH form suggests that this reaction may be in part diffusion controlled. This conjecture is borne out by the observation that the sensitivity of k(cat)/K(m) to eta(rel) decreases with increasing pH for VI, whereas this sensitivity is pH independent for I. These conclusions are further supported by the results of a kinetic investigation of the pH dependence of sulbenicillin (VII) where an alpha-SO3H replaces the alpha-COOH of VI. The strongly acidic sulfonic acid moiety of VII is fully ionized throughout nearly the entire pH range of interest, and its kinetics, as a function of pH, are very similar to those observed and calculated for the alpha-COO form of VI. Solvent deuterium kinetic isotope effects are reported for k(cat) and k(cat)/K(m) for both VI and VII.