Investigation of membrane disruption in the reaction catalyzed by cholesterol oxidase

Dye leakage experiments were undertaken to investigate the membrane disruption properties of cholesterol oxidase. Inspection of the X-ray crystal structures of cholesterol oxidase suggested that an active-site "lid" opens in order to bind substrate [Li, J., Vrielink, A., Brick, P., & Blow, D. M. (1993) Biochemistry 32, 11507-11515]. We tested whether the interaction of the putative active-site lid with the membrane was sufficiently disruptive of the membrane structure to cause leakage or lysis of the cell membrane. Vesicles (100 nm) composed of egg phosphatidylcholine, 2-palmitoyl-3-oleoyl-1-sn-phosphatidylethanolamine, and 2-palmitoyl-3-oleoyl-1-sn-phosphatidylcholine were used in this study to mimic biomembranes. To separate the effects of membrane binding from conversion of cholesterol to cholest-4-en-3-one, the active-site mutant E361Q was utilized. In the reaction catalyzed by E361Q, isomerization of the cholest-5-en-3-one intermediate is suppressed and cholest-5-en-3-one is the major product isolated. Furthermore, E361Q produces cholest-5-en-3-one 20-fold more slowly than wild type produces cholest-4-en-3-one from cholesterol. Wild-type and E361Q cholesterol oxidases bind to vesicles with an apparent K(D) of approximately 25 microM, as measured by quenching of intrinsic tryptophan fluorescence, irrespective of headgroup size and cholesterol content. Membrane disruption was measured by leakage of the encapsulated marker carboxyfluorescein. Leakage was observed with cholesterol-containing vesicles and wild-type enzyme only; the rate of leakage was dependent on the rate of cholest-4-en-3-one production. E361Q did not induce membrane disruption, regardless of vesicle type tested. Thus, binding of cholesterol oxidase to the membrane and partitioning of cholesterol into the active site does not sufficiently perturb the bilayer to cause leakage of vesicle contents. Formation of the product cholest-4-en-3-one, however, does increase membrane permeability. Expansion of the lipid bilayer upon conversion of cholesterol to cholest-4-en-3-one is the likely cause of this increased permeability.     

The importance of GLU361 position in the reaction catalyzed by cholesterol oxidase

Cholesterol oxidase stereospecifically isomerizes cholest-5-en-3-one to cholest-4-en-3-one. When the base catalyst for isomerization, Glu361, is mutated to Asp, the rate of deprotonation of cholest-5-en-3-one is not affected, but protonation of the dienolic intermediate becomes rate-limiting. This may be a consequence of the large distance between the catalytic base and carbon-6 of the intermediate in the mutant enzyme.     

Thromboembolic events in beta thalassemia major: an Italian multicenter study

Oxidation of cholesterol (1a) or pregnenolone (1b) with pyridinium dichromate (PDC) in dimethylformamide (DMF) or in dichloromethane (DCM) and pyridinium chlorochromate (PCC) in DCM provided cholest-4-en-3,6-dione (2a) or pregn-4-en-3,6,20-trione (2b). TLC monitoration of the reactions implied that cholest-5-en-3-one (3a) or pregn-5-en-3,20-dione (3b) and cholest-4-en-3-one (4a) or pregn-4-en-3,20-dione (4b) might be intermediates. Individual oxidation of 3a or 3b with PDC and PCC could give 2a or 2b, but 4a or 4b remained unchanged. Further investigation indicated that 4a or 4b was an isomerization product of 3a or 3b on silica gel TLC plate rather than really existence in the reaction mixture. These results shown steroidal 5-en-3-ones were intermediates of the transformation of steroidal 5-en-3 beta-ols to steroidal 4-en-3,6-diones oxidized by PDC and PCC.     

Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants

High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q > E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction.     

Separation of the two reactions, oxidation and isomerization, catalyzed by Streptomyces cholesterol oxidase

Site-directed mutagenesis was used to identify key amino acid residues of the cholesterol oxidase from Streptomyces sp., which catalyzes the oxidation of cholesterol and the isomerization of 5-cholesten-3-one. Eight mutant enzymes were constructed and the following amino acid substitutions were identified: N318A, N318H, E356A, E356D, H441A, H441N, N480A and N480Q. Mutants N318A and N318H retained both oxidation and isomerization activities. The mutant E356D retained oxidation but not isomerization activity. On the other hand, mutants N480A and N480Q showed no oxidation activity but retained their isomerization activities. The two catalytic reactions, oxidation and isomerization, in cholesterol oxidase were thus successfully separated. When the H441A or H441N mutation was introduced, both the oxidase and isomerase activities were completely lost. The H441, E356 and N480 residues thus appear to participate in the catalysis of cholesterol oxidase, whereas N318 does not. An analysis of the products of these mutant enzymes suggested that the previously proposed 6-hydroxylation reaction by cholesterol oxidase is actually autooxidation from 5-cholesten-3-one. Kinetic studies of the purified wild-type and mutant enzymes showed that the k(cat)/Km values for oxidation in E356D and for isomerization in N480A increased six- and threefold, respectively, over those in the wild-type. These mutational effects and the reaction mechanisms are discussed in terms of the three-dimensional structure of the enzyme constructed on the basis of homology modeling.     

The axial tyrosinate Fe3+ ligand in protocatechuate 3,4-dioxygenase influences substrate binding and product release: evidence for new reaction cycle intermediates

The essential active site Fe3+ of protocatechuate 3,4-dioxygenase [3, 4-PCD, subunit structure (alphabetaFe3+)12] is bound by axial ligands, Tyr447 (147beta) and His462 (162beta), and equatorial ligands, Tyr408 (108beta), His460 (160beta), and a solvent OH- (Wat827). Recent X-ray crystallographic studies have shown that Tyr447 is dissociated from the Fe3+ in the anaerobic 3,4-PCD complex with protocatechuate (PCA) [Orville, A. M., Lipscomb, J. D., and Ohlendorf, D. H. (1997) Biochemistry 36, 10052-10066]. The importance of Tyr447 to catalysis is investigated here by site-directed mutation of this residue to His (Y447H), the first such mutation reported for an aromatic ring cleavage dioxygenase containing Fe3+. The crystal structure of Y447H (2.1 A resolution, R-factor of 0.181) is essentially unchanged from that of the native enzyme outside of the active site region. The side chain position of His447 is stabilized by a His447(N)delta1-Pro448(O) hydrogen bond, placing the Nepsilon2 atom of His447 out of bonding distance of the iron ( approximately 4.3 A). Wat827 appears to be replaced by a CO32-, thereby retaining the overall charge neutrality and coordination number of the Fe3+ center. Quantitative metal and amino acid analysis shows that Y447H binds Fe3+ in approximately 10 of the 12 active sites of 3,4-PCD, but its kcat is nearly 600-fold lower than that of the native enzyme. Single-turnover kinetic analysis of the Y447H-catalyzed reaction reveals that slow substrate binding accounts for the decreased kcat. Three new kinetically competent intermediates in this process are revealed. Similarly, the product dissociation from Y447H is slow and occurs in two resolved steps, including a previously unreported intermediate. The final E.PCA complex (ES4) and the putative E.product complex (ESO2*) are found to have optical spectra that are indistinguishable from those of the analogous intermediates of the wild-type enzyme cycle, while all of the other observed intermediates have novel spectra. Once the E.S complex is formed, reaction with O2 is fast. These results suggest that dissociation of Tyr447 occurs during turnover of 3,4-PCD and is important in the substrate binding and product release processes. Once Tyr447 is removed from the Fe3+ in the final E.PCA complex by either dissociation or mutagenesis, the O2 attack and insertion steps proceed efficiently, suggesting that Tyr447 does not have a large role in this phase of the reaction. This study demonstrates a novel role for Tyr in a biological system and allows evaluation and refinement of the proposed Fe3+ dioxygenase mechanism.     

Mutations in the activation loop tyrosine of the oncoprotein v-Fps

Mutations were made in the activation loop tyrosine of the kinase domain of the oncoprotein v-Fps to assess the role of autophosphorylation in catalysis. Three mutant proteins, Y1073E, Y1073Q, and Y1073F, were expressed and purified as fusion proteins of glutathione-S-transferase from Escherichia coli and their catalytic properties were evaluated. Y1073E, Y1073Q, and Y1073F have k(cat) values that are reduced by 5-, 35-, and 40-fold relative to the wild-type enzyme, respectively. For all mutant enzymes, the Km values for ATP and a peptide substrate, EAEIYEAIE, are changed by 0.4-2-fold compared to the wild-type enzyme. The slopes for the plots of relative turnover versus solvent viscosity [(k(cat))eta] are 0.71 +/- 0.08, 0.10 +/- 0.06, and approximately 0 for wild type, Y1073Q, and Y1073E, respectively. These results imply that the phosphoryl transfer rate constant is reduced by 19- and 130-fold for Y1073E and Y1073Q compared to the wild-type enzyme. The dissociation constant of the substrate peptide is 1.5-2.5-fold lower for the mutants compared to wild type. The inhibition constant for EAEIFEAIE, a competitive inhibitor, is unaffected for Y1073E and raised 3-fold for Y1073Q compared to wild type. Y1073E and Y1073Q are strongly activated by free magnesium to the same extent and the apparent affinity constant for the metal is similar to that for the wild-type enzyme. The data indicate that the major role of autophosphorylation in the tyrosine kinase domain of v-Fps is to increase the rate of phosphoryl transfer without greatly affecting active-site accessibility or the local environment of the activating metal. Finally, the similar rate enhancements for phosphoryl transfer in v-Fps compared to protein kinase A [Adams et al. (1995) Biochemistry 34, 2447-2454] upon autophosphorylation suggest a conserved mechanism for communication between the activation loop and the catalytic residues of these two enzymes.     

Biological and clinicopathological features associated with hepatitis C virus type 5 infections

In previous work, we showed that o-quinones (3,5-cyclohexadiene-1,2-diones) can isomerize to p-quinone methides (4-alkyl-2,5-cyclohexadien-1-one) at rates which depend on the type of substituent at the para position [Iverson, S. L., Hu, L. Q., Vukomanovic, V., and Bolton, J. L. (1995) Chem. Res. Toxicol. 8, 537-544]. In the present investigation, we explored the mechanism of this isomerization reaction using 4-propyl-3,5-cyclohexadiene-1,2-dione (PQ) and its benzyl dideuterio analog 4-(1',1'-dideuteriopropyl)-3,5-cyclohexadiene-1, 2-dione (DPQ). The results show that the isomerization reaction is general base-catalyzed, which suggests that amino acids on proteins with basic side chains could catalyze the reaction in vivo. The Bronsted beta value was determined to be 0.23 +/- 0.02, consistent with the transfer of a proton in the rate-determining step. The rate/pH profile generated from the buffer dilution plots showed dependence on hydroxide ion concentration from pH 7.8 to 9, indicative of base catalysis. From pH 6 to 7.8, the reaction was independent of pH, suggesting that other processes compete at low buffer concentration in this pH region. Substitution of the benzyl CH2 group with CD2 dramatically slows the isomerization reaction. The kinetic deuterium isotope effect on quinone methide formation was determined by measuring the amount of quinone methide trapped as GSH conjugates from PQ compared with DPQ. The isotope effect on product formation was 5.5 +/- 0.6, 37 degrees C. These data provide further evidence that formation of these electrophilic quinone methides from o-quinones could be catalyzed by basic residues in vivo and that the reaction could be inhibited by deuterium substitution at the benzyl methylene group.     

The scope and limitations of the reaction of delta 5-steroids with mercury(II) trifluoroacetate

The effect of the C-3 substituent on the reaction of androst-5-enes with mercury(II) trifluoroacetate in dichloromethane (modified Treibs oxidation) was investigated. 3 beta-Acyloxyandrost-5-en-17-ones gave 3 beta-acyloxy-6 beta-hydroxyandrost-4-en-17-ones accompanied by 3 beta-acyloxy-6-chloromercuriandrost-5-en-17-ones. 3 beta-Acetoxy-6 beta-trifluoroacetoxyandrost-4-en-17-one and 3 beta-acetoxy-4 beta-trifluoroacetoxyandrost-5-en-17-one were revealed to be intermediates in the reaction. The formation of the chloromercury steroids indicated participation in the reaction by the solvent. With 3 alpha-acetoxyandrost-5-en-17-one as substrate, a complete reversal in the product distribution was observed. 3 beta-Haloandrost-5-en-17-ones gave mainly products that reflected SN1 substitution of the halide. 3 beta-Hydroxy- and 3 beta-trifluoroacetoxyandrost-5-en-17-ones were formed. 3 beta-Methoxyandrost-5-en-17-one afforded in nearly identical yields androst-4-ene-3,17-dione, 3 beta-methoxy-6 beta-hydroxyandrost-4-en-17-one, 3 beta-methoxy-6-chloromercuriandrost-5-en-17-one and 6 beta-hydroxyandrost-4-ene-3,17-dione while androst-5-en-17-one yielded 3 beta,6 beta-dihydroxyandrost-4-en-17-one, androst-5-ene-7,17-dione and androst-4-ene-3,17-dione. The effects of solvent and other mercury salts on the reaction were also studied. Treibs oxidation was successful in chloroform, carbon tetrachloride, and dibromomethane, but not in other solvents tested. 3 beta-Acetoxy-6-bromomercuriandrost-5-en-17-one was obtained in dibromomethane. Replacement of the reagent by mercury(II) trichloroacetate altered the intermediates formed but not the products. Mercury(II) tribromoacetate was unreactive, however.     

How enzymes control the reactivity of adenosylcobalamin: effect on coenzyme binding and catalysis of mutations in the conserved histidine-aspartate pair of glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through the formation of radical intermediates. It shares a structurally similar cobalamin-binding domain with methylcobalamin-dependent methionine synthase. In particular, both proteins contain the "DXHXXG" cobalamin-binding motif, in which the histidine provides the axial ligand to cobalt. The effects of mutating the conserved histidine and aspartate residues in methionine synthase have recently been described [Jarrett, J. T., Amaratunga, M., Drennan, C. L., Scholten, J. D., Sands, R. H., Ludwig, M. L., & Matthews, R. G. (1996) Biochemistry 35, 2464-2475]. Here, we describe how similar mutations in the "DXHXXG" motif of glutamate mutase affect coenzyme binding and catalysis in an adenosylcobalamin-dependent reaction. The mutations made in the MutS subunit of glutamate mutase were His16Gly, His16Gln, Asp14Asn, Asp14Glu, and Asp14Ala. All the mutations affect, in varying degrees, the rate of catalysis, the affinity of the protein for the coenzyme, and the coordination of cobalt. Mutations of either Asp14 or His16 decrease k(cat) by 1000-fold, and whereas cob(II)alamin accumulates as an intermediate in the wild-type enzyme, it does not accumulate in the mutants, suggesting the rate-determining step is altered. The apparent Kd for adenosylcobalamin is raised by about 50-fold when His16 is mutated and by 5-10-fold when Asp16 is mutated. There are extensive differences between the UV-visible spectra of wild-type and mutant holoenzymes, indicating that the mutant enzymes coordinate cobalt less well. Overall, the properties of these mutants differ quite markedly from those observed when similar mutations were introduced into methionine synthase.     

Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin

Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu122 and His211 on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu122 allowed assignments of the C=O stretch of protonated Glu122 in the dark state and in MI of Rho. Mutation of His211, but not of other histidine residues, affects these vibrational modes assigned to Glu122. In addition, replacements of His211 affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu122. These modes are influenced as well when Glu122 is replaced by Ala in mutant E122A but not when it is replaced by Gln in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu122 and His211.     

Mutational analysis of the role of the distal histidine and glutamine residues of prostaglandin-endoperoxide synthase-2 in peroxidase catalysis, hydroperoxide reduction, and cyclooxygenase activation

Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.     

Characterization of Glu126 and Arg144, two residues that are indispensable for substrate binding in the lactose permease of Escherichia coli

Glu126 and Arg144 in the lactose permease are indispensable for substrate binding and probably form a charge-pair [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807]. Mutants with Glu126-->Ala or Arg144-->Ala do not bind ligand or catalyze lactose accumulation, efflux, exchange, downhill lactose translocation, or lactose-induced H+ influx. In contrast, mutants with conservative mutations (Glu126-->Asp or Arg144-->Lys) exhibit drastically different phenotypes. Arg144-->Lys permease accumulates lactose slowly to low levels, but does not bind ligand or catalyze equilibrium exchange, efflux, or lactose-induced H+ influx. In contrast, Glu126-->Asp permease catalyzes lactose accumulation and lactose-induced H+ influx to wild-type levels, but at significantly lower rates. Surprisingly, however, no significant exchange or efflux activity is observed. Glu126-->Asp permease exhibits about a 6-fold increase in the Km for active transport relative to wild-type permease with a comparable Vmax. Direct binding assays using flow dialysis demonstrate that mutant Glu126-->Asp binds p-nitrophenyl-alpha,D-galactopyranoside. Indirect binding assays utilizing substrate protection against [14C]-N-ethylmaleimide labeling of single-Cys148 permease reveal an apparent Kd of 3-5 mM for lactose and 15-20 microM for beta, D-galactopyranosyl-1-thio-beta,D-galactopyranoside (TDG). The affinity of Glu126-->Asp/Cys148 permease for lactose is markedly decreased (Kd > 80 mM), while TDG affinity is altered to a much lesser extent (Kd ca. 80 microM). The results extend the conclusion that a carboxylate at position 126 and a guanidinium group at position 144 are irreplaceable for substrate binding and support the idea that Arg144 plays a major role in substrate specificity.     

Reaction mechanism of fructose-2,6-bisphosphatase. A mutation of nucleophilic catalyst, histidine 256, induces an alteration in the reaction pathway

A bifunctional enzyme, fructose-6-phosphate,2-kinase/fructose 2, 6-bisphosphatase (Fru-6-P,2-kinase/Fru-2,6-Pase), catalyzes synthesis and degradation of fructose 2,6-bisphosphate (Fru-2,6-P2). Previously, the rat liver Fru-2,6-Pase reaction (Fru-2,6-P2 --> Fru-6-P + Pi) has been shown to proceed via a phosphoenzyme intermediate with His258 phosphorylated, and mutation of the histidine to alanine resulted in complete loss of activity (Tauler, A., Lin, K., and Pilkis, S. J. (1990) J. Biol. Chem. 265, 15617-15622). In the present study, it is shown that mutation of the corresponding histidine (His256) of the rat testis enzyme decreases activity by less than a factor of 10 with a kcat of 17% compared with the wild type enzyme. Mutation of His390 (in close proximity to His256) to Ala results in a kcat of 12.5% compared with the wild type enzyme. Attempts to detect a phosphohistidine intermediate with the H256A mutant enzyme were unsuccessful, but the phosphoenzyme is detected in the wild type, H390A, R255A, R305S, and E325A mutant enzymes. Data demonstrate that the mutation of His256 induces a change in the phosphatase hydrolytic reaction mechanism. Elimination of the nucleophilic catalyst, H256A, results in a change in mechanism. In the H256A mutant enzyme, His390 likely acts as a general base to activate water for direct hydrolysis of the 2-phosphate of Fru-2,6-P2. Mutation of Arg255 and Arg305 suggests that the arginines probably have a role in neutralizing excess charge on the 2-phosphate and polarizing the phosphoryl for subsequent transfer to either His256 or water. The role of Glu325 is less certain, but it may serve as a general acid, protonating the leaving 2-hydroxyl of Fru-2,6-P2.     

Catalytic domain of phosphoinositide-specific phospholipase C (PLC). Mutational analysis of residues within the active site and hydrophobic ridge of plcdelta1

Structural studies of phospholipase C delta1 (PLCdelta1) in complexes with the inositol-lipid headgroup and calcium identified residues within the catalytic domain that could be involved in substrate recognition, calcium binding, and catalysis. In addition, the structure of the PLCdelta1 catalytic domain revealed a cluster of hydrophobic residues at the rim of the active site opening (hydrophobic ridge). To assess a role of each of these residues, we have expressed, purified, and characterized enzymes with the point mutations of putative active site residues (His311, Asn312, Glu341, Asp343, His356, Glu390, Lys438, Lys440, Ser522, Arg549, and Tyr551) and residues from the hydrophobic ridge (Leu320, Phe360, and Trp555). The replacements of most active site residues by alanine resulted in a great reduction (1,000-200,000-fold) of PLC activity analyzed in an inositol lipid/sodium cholate mixed micelle assay. Measurements of the enzyme activity toward phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4, 5-bis-phosphate (PIP2) identified Ser522, Lys438, and Arg549 as important for preferential hydrolysis of polyphosphoinositides, whereas replacement of Lys440 selectively affected only hydrolysis of PIP2. When PLC activity was analyzed at different calcium concentrations, substitutions of Asn312, Glu390, Glu341, and Asp343 resulted in a shift toward higher calcium concentrations required for PIP2 hydrolysis, suggesting that all these residues contribute toward Ca2+ binding. Mutational analysis also confirmed the importance of His311 ( approximately 20,000-fold reduction) and His356 ( approximately 6,000-fold reduction) for the catalysis. Mutations within the hydrophobic ridge, which had little effect on PIP2 hydrolysis in the mixed-micelles, resulted in an enzyme that was less dependent on the surface pressure when analyzed in a monolayer. This systematic mutational analysis provides further insights into the structural basis for the substrate specificity, requirement for Ca2+ ion, catalysis, and surface pressure/activity dependence, with general implications for eukaryotic phosphoinositide-specific PLCs.     

Roles of histidine 752 and glutamate 699 in the pH dependence of mouse band 3 protein-mediated anion transport

In the accompanying paper we have shown that four different histidine residues are involved in the maintenance of mouse band 3 in a state in which it is able to execute its anion transport function. Here we focus on the functional significance of His 752 and demonstrate that this residue, together with Glu 699, plays a key role in the control of pH dependence of Cl- transport. Mouse band 3-encoding cRNA was expressed in Xenopus oocytes, and band 3-mediated Cl- transport was measured at zero membrane potential over the pH range 6.0-9.2. Transport decreased with increasing H+ concentration and was governed by a single pK of 5.8. After correction for temperature differences, this result agrees well with measurements in erythrocyte ghosts of Cl- flux by Funder and Wieth [Funder, J., & Wieth, J. O. (1976) J. Physiol. 262, 679-698] and our own determinations by 35Cl NMR spectroscopy of Cl- exchange between the substrate binding site and the medium. After mutation of either Glu 699 to Asp or of His 752 to Ser, the maximal rate of transport is reduced and the rate of anion exchange is now governed by a single pK of about 6.8-6.9. This suggests that the formation of a hydrogen bond between His 752 and Glu 699 is essential for the decrease of band 3-mediated Cl- transport at low pH. We suggest that in the wild type band 3 both the decrease of the chloride exchange between the medium and the substrate binding site and the inhibition of chloride translocation across the membrane are dominated by a common rate-limiting step and that this step involves hydrogen bond formation between Glu 699 and His 752.     

Multiple isotope effects as a probe of proton and hydride transfer in the 6-phosphogluconate dehydrogenase reaction

Primary solvent deuterium, primary substrate deuterium, multiple solvent deuterium/substrate deuterium, and multiple solvent deuterium/13C isotope effects on V/K6PG have been measured for the Candida utilis and sheep liver 6-phosphogluconate dehydrogenases (6PGDH). Proton inventory data suggest the presence of a significant medium effect in a step preceding hydride transfer and the presence of a kinetic solvent deuterium isotope effect on hydride transfer. Multiple isotope effect data confirm the presence of multiple solvent deuterium sensitive steps, likely including a conformational change preceding hydride transfer, hydride transfer, and decarboxylation.     

Role of lysine-57 in the catalytic activities of Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg protein)

The Escherichia coli Fpg protein is involved in the repair of oxidized residues. We examined, by targeted mutagenesis, the effect of the conserved lysine residue at position 57 upon the various catalytic activities of the Fpg protein. Mutant Fpg protein with Lys-57-->Gly (K57G) had dramatically reduced DNA glycosylase activity for the excision of 7,8-dihydro-8-oxo-guanine (8-oxoG). While wild type Fpg protein cleaved 8-oxoG/C DNA with a specificity constant ( k cat/ K M) of 0.11/(nM@min), K57G cleaved the same DNA 55-fold less efficiently. FpgK57G was poorly effective in the formation of Schiff base complex with 8-oxoG/C DNA. The efficiency in the binding of 8-oxoG/C DNA duplex for K57G mutant was decreased 16-fold. The substitution of Lys-57 for another basic amino acid Arg (K57R) had a slight effect on the 8-oxoG-DNA glycosylase activity and Schiff base formation. The DNA glycosylase activities of FpgK57G and FpgK57R using 2,6-diamino-4-hydroxy-5N-methylformamidopyrimidine residues as substrate were comparable to that of wild type Fpg. In vivo, the mutant K57G, in contrast to the mutant K57R and wild type Fpg, only partially restored the ability to prevent spontaneously induced transitions G/C-->T/A in E.coli BH990 ( fpg mutY ) cells. These results suggest an important role for Lys-57 in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo.     

Studies on the receptors to 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol in sow nasal mucosa

The presence of receptors to the "boar taint" pheromones 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol has been demonstrated in sow olfactory mucosa. Binding studies indicated that a sufficiently low concentration of olfactory tissue homogenate exhibited saturation of binding of 5alpha-androst-16-en-3-one, and this was of high affinity compared with control tissues of non-olfactory and heated olfactory tissues. Analysis of receptor binding of 5alpha-androst-16-en-3-one gave a value for the affinity constant (Ka) of approx. 8.3-10(8) M-1 and the value for the molar concentration of binding sites (n[M]) was approx. 3.3 pmol/mg protein. Almost identical values of Ka and n [M] were obtained when receptor binding of 5alpha-[5alpha-3H]androst-16-en-3alpha-ol was investigated (Ka 8.4-10(8) M-1; n [M] 3.7 pmol/mg protein). This suggests that the same receptor binds both 5alpha-androst-16-en-3-one and 5alpha-androst-16-en-3alpha-ol with equally high affinity. In a preliminary investigation to establish the specificity of the receptor, the binding of 17beta-hydroxy-5alpha-androstan-3-one was assayed; this steroid is odourless but has a similar structure except in ring D to 5alpha-androst-16-en-3-one. Binding was of the low affinity, non-specific type only, indicating that the sow olfactory receptors are not sensitive to this androgen.     

Cholesterol and oxygenated cholesterol concentrations are markedly elevated in peripheral tissue but not in brain from mice with the Niemann-Pick type C phenotype

Niemann-Pick disease type C (NP-C) is a rare genetic disorder characterized by progressive neurodegeneration, frequent developmental delay and early death. Tissues of affected individuals accumulate large quantities of free cholesterol in lysosomes. Because cytotoxic oxygenated derivatives of cholesterol are known to form readily when cholesterol concentrations are elevated, we searched for these compounds in liver, kidney, spleen and brain from mice with the NP-C phenotype. In order of abundance, we identified 7 alpha- and 7 beta-hydroxycholesterol, 5 alpha, 6 alpha-epoxycholestan-3 beta-ol, 4 beta-hydroxycholesterol, cholest-4-en-3 beta, 7 alpha-diol and cholest-4-en-3 beta, 6 beta-diol in most tissue samples. Cholesterol concentrations in affected mice were increased 3-fold in kidney and 7- to 8-fold in spleen and liver compared to controls (all p < 0.001) but were unchanged in brain. Although oxysterol levels were markedly elevated in nonbrain tissue, the oxysterol and cholesterol concentrations increased proportionally so that oxysterols expressed as percentage of total sterols were the same for all animals (0.34 +/- 0.19% averaged over all organs in affected animals vs 0.40 +/- 0.42% in control mice). In contrast to peripheral tissue, we could not detect any increase in either absolute or relative oxysterol levels in the brains of affected and control mice (49 +/- 61 vs 53 +/- 43 micrograms/g wet weight and 0.45 +/- 0.52 vs 0.47 +/- 0.37%, respectively). Thus, brain sterols are normal in NP-C mice and it is unlikely that an accumulation of cytotoxic oxygenated derivatives of cholesterol could account for the progressive neuropathology seen in the disease.