Redox potentials of trinuclear μ-oxo ruthenium acetate clusters with N-heterocyclic ligands

The trinuclear clusters of general composition [Ru₃O(OOCCH₃)₆(N-Het)₃], where N-Het=pyridine and pyrazine derivatives, exhibit a series of reversible waves in the range of −1.8 to 2.4 V versus SHE, in acetonitrile, ascribed to the successive [cluster]^{−2/−1/0/+1/+2/+3} redox couples. The redox potentials decrease with the pK_a of the N-heterocyclic ligands according to the equations E°(+3/+2)= 2.24−0.023 pK_a; E°(+2/+1)=1.34−0.029 pK_a; E°(+1/0)=0.36−0.039 pK_a and E°(0/−1)=−0.68− 0.074 pK_a. The dependence is greater at lower oxidation states, reflecting the role of π-backbonding in the complexes.     

On the pH-dependence of the light-induced hydrogen ion gradient in spinach chloroplasts

The dependence of the light-induced H⁺ gradient in chloroplasts (ΔpH) on external pH was examined using the distribution of aniline, an amine of low pK_a. ΔpH was essentially independent of pH over the range of 7–8. It was previously reported that ΔpH, determined from the distribution of relatively polar amines of high pK_a, decreased as the pH was lowered below 8. It is suggested that, in the case of amines of high pK_a, ΔpH values determined at low external pH values are too low because the permeability of chloroplasts to the amine cation relative to that of the unprotonated form may be significant.     

The structure of carbonyl horseradish peroxidase: spectroscopic and kinetic characterization of the carbon monoxide complexes of His-42 → Leu and Arg-38 → Leu mutants

Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the involvement of two key distal heme cavity residues, histidine 42 and arginine 38, in the formation and structure of the carbon monoxide complex of HRPC (carbonyl HRPC). The rates of CO binding to the wild-type glycosylated and non-glycosylated recombinant (HRPC*) ferrous enzymes were essentially identical and exhibited the same pH dependence with pK _as at 7.4 and 4.0. Data obtained with the His-42?→?Leu [(H42L)HRPC*)] and Arg-38?→?Leu [(R38L)HRPC*] mutants allowed the pK _a at 7.4 in ferrous HRPC to be assigned to His-42. The infra-red and electronic absorption spectra of HRPC-CO, HRPC*-CO, (R38L)HRPC*-CO and (H42L)HRPC*-CO have been investigated over the pH range 3.0–10.0. HRPC*-CO exhibited two ν?(CO) bands at 1934?cm^–1 and 1905?cm^–1 whose relative intensity changed with pH, showing an acidic and a basic pK _a as previously reported for HRPC [IE Holzbaur; AM English, AA Ismail (1996) J Am Chem Soc 118?:?3354–3359]. (H42L)HRPC*-CO and (R38L)HRPC*-CO exhibited single infra-red bands at 1924.2?cm^–1 (pH?7.0) and 1941.5?cm^–1 (pH?5.0) respectively. Acidic and alkaline pK _as were determined from shifts in the infra-red frequencies and by UV-visible spectrophotometry at the Söret maxima. (H42L)HRPC*-CO exhibited a pK _a at ～pH?4.0 but no alkaline pK _a. (R38L)HRPC*-CO exhibited a single pK _a at pH?6.5. Shifts of 2–3?cm^–1 in ν?(CO) with (H42L)HRPC*-CO in D₂O show that a distal residue is H-bonding to the CO in this variant at both pD?7.5 and 3.9. However, with (R38L)HRPC*-CO, only a small shift of the ν?(CO) band was observed at pD?5.5. The results are consistent with the involvement of Arg-38 in H-bonding to the CO ligand in HRPC and with His-42 modulating the distribution of carbonyl HRPC conformers below pH?8.7. These data are discussed in terms of the importance of distal pocket polarity in HRPC. It is concluded that His-42 can have a pK _a between 4.0 and 8.7 depending on its environment and the nature of the distal ligand at position 38. This enables His-42 to carry out multiple functions during the catalytic cycle of HRPC.     

The One-Electron Reduction Potential of 3-Amino-1, 2, 4-benzotriazine 1, 4-dioxide (Tirapazamine): A Hypoxia-Selective Bioreductive Drug

The one-electron reduction potential of 3-amino-l, 2, 4-benzotriazine 1, 4-dioxide, tirapazamine (SR 4233) in aqueous solution has been determined by pulse radiol-ysis. Reversible electron transfer was achieved between radiolytically-generated one-electron reduced radicals of tirapazamine (T), and quinones or benzyl viologen as redox standards. The reduction potential E_m7(T/T^±) was -0.45 ± 0.01 V vs. NHE at pH 7. From the pH dependence of the reduction potential, pK_a = 5.6 ± 0.2 was estimated for the tirapazamine radical, a value similar to the pK_a determined by other methods.     

Carboxyl pKa Values and Acid Denaturation of BBL

The protein BBL undergoes structural transitions and acid denaturation between pH 1.2 and 8.0. Using NMR spectroscopy, we measured the pK_a values of all the carboxylic residues in this pH range. We employed ¹³C direct-detection two-dimensional IPAP (in-phase antiphase) CACO NMR spectroscopy to monitor the ionization state of different carboxylic groups and demonstrated its advantages over other NMR techniques in measuring pK_a values of carboxylic residues. The two residues Glu161 and Asp162 had significantly lowered pK_a values, showing that these residues are involved in a network of stabilizing electrostatic interactions, as is His166. The other carboxylates had unperturbed values. The pH dependence of the free energy of denaturation was described quantitatively by the ionizations of those three residues of perturbed pK_a, and, using thermodynamic cycles, we could calculate their pK_as in the native and denatured states as well as the equilibrium constants for denaturation of the different protonation states. We also measured ¹³C^α chemical shifts of individual residues as a function of pH. These shifts sense structural transitions rather than ionizations, and they titrated with pH consistent with the change in equilibrium constant for denaturation. Kinetic measurements of the folding of BBL E161Q indicated that, at pH 7, the stabilizing interactions with Glu161 are formed mainly in the transition state. We also found that local interactions still exist in the acid-denatured state of BBL, which attenuate somewhat the flexibility of the acid-denatured state.     

Aggregation of ferrihaems. Dimerization and protolytic equilibria of protoferrihaem and deuteroferrihaem in aqueous solution

1. The absorption spectra of deutero- and proto-ferrihaem in aqueous solution at 25°C show marked changes with concentration and pH in the Soret band region. Quantitative studies of these phenomena imply that they are associated with ferrihaem dimerization and with protolytic equilibria involving monomeric (M) and dimeric (D) ferrihaem species according to the scheme: [Formula: see text] 2. For deuteroferrihaem we obtain K=1.9×10⁻², pK_a(M)=7.1, pK_a(D)=7.4. Protoferrihaem has a much higher dimerization constant, K=4.5 and pK_a(D)=7.5 (pK_a(M) is not accessible). 3. Possible structural relationships between monomeric and dimeric ferrihaem species in solution are discussed in relation to recent work on the oxo-bridged nature of crystalline ferrihaem dimers.     

Protonation of kanamycin A: Detailing of thermodynamics and protonation sites assignment

Protonation of an aminoglycoside antibiotic kanamycin A sulfate was studied by potentiometric titrations at variable ionic strength, sulfate concentration and temperature. From these results the association constants of differently protonated forms of kanamycin A with sulfate and enthalpy changes for protonation of each amino group were determined. The protonation of all amino groups of kanamycin A is exothermic, but the protonation enthalpy does not correlate with basicity as in a case of simple polyamines. The sites of stepwise protonation of kanamycin A have been assigned by analysis of ¹H-¹³C-HSQC spectra at variable pH in D₂O. Plots of chemical shifts for each H and C atom of kanamycin A vs. pH were fitted to the theoretical equation relating them to pK_a values of ionogenic groups and it was observed that changes in chemical shifts of all atoms in ring C were controlled by ionization of a single amino group with pK_a 7.98, in ring B by ionization of two amino groups with pK_a 6.61 and 8.54, but in ring A all atoms felt ionization of one group with pK_a 9.19 and some atoms felt ionization of a second group with pK_a 6.51, which therefore should belong to amino group at C3 in ring B positioned closer to the ring A while higher pK_a 8.54 can be assigned to the group at C1. This resolves the previously existed uncertainty in assignment of protonation sites in rings B and C.     

Heme-linked ionization and chloride binding in intestinal peroxidase and lactoperoxidase.

Hog intestinal peroxidase and bovine lactoperoxidase exhibited similar spectral shifts upon pH alteration. From spectrophotometric titrations, it was found that there are hemelinked ionizations of pK_a = 4.75 in intestinal peroxidase and pK_a = 3.5 in lactoperoxidase. The apparent pK_a (pK_a′) increased with the increase in chloride concentration. The pK_a′ vs log[Cl^?] plots showed that the chloride forms complex with the acid forms of these enzymes with a dissociation constant (pK = 2.7). Although the dissociation constant (K_d) of the peroxidase-cyanide complexes is nearly independent of pH, cyanide competed with chloride in the acidic pH region. The slopes of logK_d vs log[Cl^?] were 1.0 for intestinal peroxidase and 0.5 for lactoperoxidase. The reaction of hydrogen peroxide with these peroxidases was also affected by chloride, similarly as the reaction with cyanide was. The results were explained by assuming that protonation occurs at the distal base and destroys the hydrogen bond between the base and a water molecule at the sixth coordinate position of the heme iron.     

Ionisations within a subtilisin–glyoxal inhibitor complex

Z-Ala-Pro-Phe-glyoxal (where Z is benzyloxycarbonyl) has been shown to be a competitive inhibitor of subtilisin with a K_i=2.3±0.2 μM at pH 7.0 and 25 °C. Using Z-Ala-Pro-[2-¹³C]Phe-glyoxal we have detected a signal at 107.3 ppm by ¹³C NMR, which we assign to the tetrahedral adduct formed between the hydroxy group of serine-195 and the ¹³C-enriched keto-carbon of the inhibitor. The chemical shift of this signal is pH independent from pH 4.2 to 7.0 and we conclude that the oxyanion pK_a<3. This is the first observation of oxyanion formation in a reversible subtilisin–inhibitor complex. The inhibitor is bound as a hemiketal which is in slow exchange with the free inhibitor. Inhibitor binding depends on a pK_a of ～6.5 in the free enzyme and on a pK_a<3.0 when the inhibitor is bound to subtilisin. Protonation of the oxyanion promotes the disassociation of the inhibitor. We show that oxyanion formation cannot be rate limiting during catalysis and that subtilisin stabilises the oxyanion by at least 45.1 kJ mol^?1. We conclude that if the energy required for oxyanion stabilisation is utilised as binding energy in drug design it should make a significant contribution to inhibitor potency.     

Interaction of Aplysia brasiliana Myoglobin with Mn2+ and Cu2+ ions

Myoglobin of Aplysia brasiliana (MbApB) has been recently purified and characterized and it was shown that the amino acid content is quite different from other myoglobins. A large number of aromatic residues was observed together with the existence of a unique histidine at the proximal heme position. Because of the numerous differences in the amino acid sequence between MbApB and whale myoglobin, it was interesting to investigate the interaction of metal ions like Cu²⁺ and Mn²⁺ with MbApB. In the present work Cu²⁺ complexes with Met-MbApB were studied and show a pH transition between different forms of coordination as revealed by EPR measurements. At high pH the EPR spectrum shows the coordination of the metal to at least four nitrogens from ϵ-NH₃ lysine residues. At lower pH in the range 6.0–9.0 the copper binding site shows a pK change of some of the residues involved in metal coordination. Addition of one equivalent Cu²⁺ per protein does not alter the iron EPR signal. The manganese ion has one binding site in MbApB and a binding constant K_a = ( 11.5 ± 0.8) 10³M⁻¹. The binding of Cu²⁺ to MbApB is stronger than Mn²⁺, K_a^Cu2+ >K_a^Mn2+.     

Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Pulse radiolysis studies of antitumor quinones: Radical lifetimes,reactivity with oxygen,and one-electron reduction potentials

The formation of semiquinone free radicals from antitumor drugs has been studied by pulse radiolysis. The semiquinone free radicals are reactive and have short half-lives in aqueous media under anaerobic conditions. The half-lives of the radicals formed from adriamycin, mitomycin C, and 2,5-diaziridinyl-3,6-bis(carboethoxy)amine-1,4-benzoquinone (AZQ) are 50,100, and 200 μs, respectively. The mean diffusion distance of the semiquinone free radical is less than 0.6 μm. In the presence of molecular oxygen the half-life of the semiquinone free radical is shortened. Adriamycin semiquinone reacts rapidly with oxygen, k = 4.4 × 10⁷m^?1s^?1. In air-saturated buffer the half-life of adriamycin semiquinone radical can be calculated to be 8 μs with a mean diffusion distance of less than 0.1 μm. If the half-lives in buffer are comparable to those within a cell, semiquinone free radicals must be generated close to the site at which they produce a biological effect. One-electron reduction potentials (E₇¹) were determined and were AZQ, ?168 mV, adrenochrome, ?253 mV, mitomycin C, ?271 mV, adriamycin, ?292 mV, daunomycin, ?305 mV, and anthracenedione, ?348 mV. Enzymatic one-electron reduction of these antitumor quinones by NADPH-cytochrome P-450 reductase increased at more positive values of quinone E₇¹.     

Isocitrate lyase from Pseudomonas indigofera: pH dependence of catalysis and binding of substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lysase from Pseudomonas indigofera was dependent on two dissociable groups (pK_a's of 6.9 and 8.6). The pH dependence of the pK_i for succinate, a product of isocitrate cleavage, implied that a dissociable group (pK_a of 6.0) on the enzyme functions in binding succinate. The pK_i's for maleate and itaconate (succinate analogs) were similarly pH dependent. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, was pH independent. In contrast the pK_i's of the four-carbon dicarboxylic acid inhibitors, fumarate and meso-tartrate, both of which affect the glyoxylate site, were dependent on a dissociable group on the enzyme-inhibitor complex. Comparison of the pH dependence of the pK_m for isocitrate and the pK_i for succinate (and succinate analogs) indicated that the binding of isocitrate was dependent on an acidic dissociable group on the enzyme (pK_a of 5.8). The pH dependence of the pK_i for homoisocitrate was similar. In addition the K_i for succinate and K_m for isocitrate were dependent upon Mg²⁺ concentration. Inhibition by phosphoenolpyruvate, which binds to the succinate site and may regulate isocitrate lyase from P. indigofera, was twice as pH dependent as that for succinate. Two dissociable groups, one on the enzyme (pK_a of 5.8) and one on phosphoenolpyruvate (pK_a of 6.35), contributed to the pH dependence observed with phosphoenolpyruvate.     

Relations between extraction protocols for activated sludge extracellular polymeric substances (EPS) and complexation properties of Pb and Cd with EPS: Part II. Consequences of EPS extraction methods on Pb2+ and Cd2+ complexation

To study the effect of extraction protocols on extracellular polymeric substances (EPS) metal binding ability, EPS from two activated sludges were extracted by eight extraction protocols: three chemical treatments, four physical treatments and a control. Two pK_a, for each EPS, were determined: pK_a1 may be specific for carboxyl and phosphoric and pK_a2 may be attributed to phenolic and amino functional groups according to EPS composition and IR spectra. EPS pK_a values could be affected by the presence of extraction reagents and/or the modifications of EPS by extraction reagents.Complexation study performed at pH 7 by a polarographic method has always showed a greater affinity of EPS for Pb²⁺ than for Cd²⁺. The complexation properties of EPS extracted by chemical methods were greatly modified. Concerning EPS extracted by physical methods, their complexation properties were close except for EPS obtained by heating. Standardized extraction methods must be established as a function of the aims of the EPS study.     

Alterations in phospholipid-dependent (Na++K+)-ATPase activity due to lipid fluidity: Effects of cholesterol and Mg2+

The (Na⁺+K⁺)-activated, Mg²⁺-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble from depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na⁺+K⁺)-ATPase in its pH optimum being around 7.0 showing optimal activity at Mg²⁺: ATP mol ratios of approximately 1 and a K_m value for ATP of 0.4 mM.Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg²⁺: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 °C, With activation energy (E_a) values of 13–15 kcal/mol above this temperature and 30–35 kcal below it. A further discontinuity was also found at 8.0 °C and the E_a below this was very high (> 100 kcal/mol).Incresed Mg²⁺ concentrations at Mg²⁺: ATP ratios in excess of 1:1 inhibited the (Na⁺+K⁺)-ATPase activity and also abolished the discontinuities in the Arrhenius plots.The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na⁺+K⁺)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20°C and E_a values of 22 and 68kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg²⁺-ATPase normally showed a linear Arrhenius plot with an E_a of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg²⁺-ATPase activity, which now also showed a discontinuity at 20 °C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the K_m for ATP.Since both of cholesterol and Mg²⁺ are know to alter the effects of temperature on the fluidity of phospholipids the above result are discussed in this context.     

3,5-Di-tert-butyl-4-hydroxybenzylidenemalononitrile. Effects of pH on its binding to liposomes and evidence for formation of a ternary complex with valinomycin and potassium ion

1. The properties of 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF 6847) were studied chemically and spectroscopically. Two molecular species of SF6847 were identified: the undissociated form (SFH; ?₃₆₃, 10 mM^?1) and the dissociated form (SF^?; ?₄₅₄, 35 mM^?1). The pK_a value of the molecule was determined to be 6.9.2. On the basis of these properties the interactions of SF6847 with liposomes and valinomycin · K⁺ were studied. The partition constants of SFH (Kⁿ_p and SF^? (K^?_p) to liposomes were determined separately; Kⁿ_p was 56 mM^?1 and was independent of the pH of the medium, whereas K^?_p dependend greatly on the pH, being 1.2 mM^?1 at pH 7.0 and 2.9 mM^?1 at pH 8.0. Using these values, the partition constant of total SF6847 (K_p) was calculated and found to be essentially the same as that calculated from the kinetics of proton uptake. It was concluded that the amount of SF^? bound to liposomes is rate limiting for proton uptake.3. The effects of membrane potential on partition constants were studied. The K^?_p decreased greatly upon generation of a membrane potential negative inside the liposomes but increased upon generation of a membrane potential positive inside the liposomes.4. The interaction of SF6847 with valinomycin in aqueous solution and in liposomes was demonstrated only in the presence of potassium ion. Potassium ion could not be replaced by sodium ion. Evidence was obtained for the formation of the ternary complex valinomycin · K⁺ · SF^? in liposomes and in hexane. It was concluded that SF^? became more soluble in the liposomal membranes on formation of this ternary complex. All these results support our proposed mechanism for the proton uptake cycle (Yamaguchi, A. and Anraku, Y. (1978) Biochim. Biophys. Acta 501, 136–149).     

Isocitrate lyase from Neurospora crassa: pH dependence of catalysis and interaction with substrates and inhibitors.

The maximal velocity, V, for isocitrate cleavage by isocitrate lyase from Neurospora crassa is dependent on two dissociable groups with pK_a values of 6.1 and 8.6. A dissociable group with a pK_a of 8.5 on the enzyme-substrate complex affects the pK_m for isocitrate. The pK_i for homoisocitrate is affected in a like manner. The pH dependence of the pK_i's for succinate, a product of isocitrate cleavage, and the succinate analog maleate is similar to the pH dependence of the pK_m of isocitrate below pH 7.3, but is markedly different above this pH. Both the K_m for isocitrate and the K_i for succinate were dependent upon Mg²⁺ concentration. The pK_i for oxalate, an analog of glyoxylate which is also a product of isocitrate cleavage, is dependent on a group with a pK_a of 6.8 on the enzyme-inhibitor complex. The pH dependence of the pK_i for phosphoenolpyruvate, which binds to the succinate site, suggests that it is dependent on two dissociable groups, one on phosphoenolpyruvate and one, by analogy to the pK_m for isocitrate, on the enzyme-glyoxylate-inhibitor complex.     

Effects of 2-deoxy-d-glucose,amiloride, vasopressin,and ouabain on active conductance andE Na in the toad bladder

Summary The effects of various agents on active sodium transport were studied in the toad bladder in terms of the equivalent circuit comprising an active conductanceK ^a, an electromotive forceE _Na, and a parallel passive conductanceK ^p. For agents which affectK ^a, but notE _Na orK ^p, the inverse slope of the plot of total conductance against short-circuit currentI ₀ evaluatesE _Na, and the intercept representsK ^p. Studies employing 5×10^–7 m amiloride to depressK ^a indicate a changingE _Na, invalidating the use of the slope technique with this agent. An alternative suitable technique employs 10^–5 m amiloride, which reducesI ₀ reversibly to near zero without effect onK ^p. Despite curvilinearity of the -I₀ plot under these conditions,K ^p may therefore be estimated fairly precisely from the residual conductance. It then becomes possible to follow the dynamic behavior ofK ^a andE _Na (in the absence of 10^–5 m amiloride) by frequent measurements of andI ₀, utilizing the relationshipsK ^a=K-K ^p, andK _Na=I _O/(K-K ^p). 2-deoxy-d-glucose (7.5×10^–3 m) depressedK ^a without affectingE _Na. Amiloride (5×10^–7 m) depressedK ^a and enhancedE _Na. Vasopressin (100 mU/ml) enhancedK ^a markedly and depressedE _Na slightly. Ouabain (10^–4 m) depressed bothK ^a andE _Na. All of the above effects were noted promptly;K ^p was unaffected. The electromotive force of Na transportE _Na appears not to be a pure energetic parameter, but to reflect kinetic factors as well, in accordance with thermodynamic considerations.     

Functional tuning of the catalytic residue pKa in a de novo designed esterase

AlleyCatE is a de novo designed esterase that can be allosterically regulated by calcium ions. This artificial enzyme has been shown to hydrolyze p‐nitrophenyl acetate (pNPA) and 4‐nitrophenyl‐(2‐phenyl)‐propanoate (pNPP) with high catalytic efficiency. AlleyCatE was created by introducing a single‐histidine residue (His¹⁴⁴) into a hydrophobic pocket of calmodulin. In this work, we explore the determinants of catalytic properties of AlleyCatE. We obtained the pK_a value of the catalytic histidine using experimental measurements by NMR and pH rate profile and compared these values to those predicted from electrostatics pK_a calculations (from both empirical and continuum electrostatics calculations). Surprisingly, the pK_a value of the catalytic histidine inside the hydrophobic pocket of calmodulin is elevated as compared to the model compound pK_a value of this residue in water. We determined that a short‐range favorable interaction with Glu¹²⁷ contributes to the elevated pK_a of His¹⁴⁴. We have rationally modulated local electrostatic potential in AlleyCatE to decrease the pK_a of its active nucleophile, His¹⁴⁴, by 0.7 units. As a direct result of the decrease in the His¹⁴⁴ pK_a value, catalytic efficiency of the enzyme increased by 45% at pH 6. This work shows that a series of simple NMR experiments that can be performed using low field spectrometers, combined with straightforward computational analysis, provide rapid and accurate guidance to rationally improve catalytic efficiency of histidine‐promoted catalysis. Proteins 2017; 85:1656–1665. © 2017 Wiley Periodicals, Inc.     

Differentiation of various types of biological oxidation of nitrogen in organic compounds

The various types of nitrogen which occur in organic compounds and which are susceptible to biological oxidation are clearly divided into groups depending upon the pK_a, of the constituent nitrogen. The enzymatic processes which give rise to the N-oxidation products are reviewed by a consideration of species differences, age of animal, pH optima, influence of inducing agents, inhibitors and microsomal pretreatments, as well as the stereochemistry of the nitrogen atom.From the data collected, a concept is developed which suggests that all basic amines (group I) are oxidised by a flavine adenine nucleotide (FAD)-dependent enzyme system, whereas non-basic nitrogen-containing compounds (group III) are oxidised by a cytochrome P450-dependent system.It is further suggested that compounds of intermediary pK_a,i.e. between 1 and 7 (group II), may be substrates for both enzyme systems, which would yield the same products, but by different processes. The extent to which N-oxidation occurs in a species would therefore depend on the pK_a of the substrate and the amounts and ratio of the two enzymes present, a lower pK_a favouring oxidation by the cytochrome P450 system and a higher pK_a favouring oxidation by the FAD system.In a similar manner, it is suggested that the oxidation of aromatic heterocyclic amines depends upon the pK_a of the nitrogen, compounds having a low pK_a being preferentially metabolised by nitrogen oxidation.