Interaction of monodispersed and micellar phospholipids with an Agkistrodon halys blomhoffii phospholipase A2, in which the alpha-amino group had been modified to an alpha-keto group

The pH dependence of the binding constant of Ca2+ to a phospholipase A2 of Agkistrodon halys blomhoffii, in which the alpha-amino group had been selectively modified to an alpha-keto group, was studied at 25 degrees C and ionic strength 0.1 by the tryptophyl fluorescence method. The dependence was compared with the results for the intact enzyme (Ikeda et al. (1981) J. Biochem. 90, 1125-1130). The pH-dependence curve could be well interpreted in terms of the participation of the two ionizable groups Asp 49 and His 48, with pK values of 4.70 and 6.69, respectively. These values were slightly different from the respective pK values for the intact enzyme, 5.15 and 6.45. Ca2+ binding to the intact enzyme involves the participation of an additional ionizable group with a pK value of 7.30, which was thus assigned as alpha-amino group. The pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) to the alpha-NH2-modified enzyme was studied at 25 degrees C and ionic strength 0.1 by the aromatic circular dichroism (CD) method. The pH-dependence curve for the modified apoenzyme was interpreted as reflecting the participation of a single ionizable group with a pK value of 4.7, which was assigned to Asp 49 (to which a Ca2+ ion can coordinate) since the curve for the Ca2+ complex lacked this transition: the binding constant was independent of pH. The pH-dependence curves for the intact apoenzyme and its Ca2+ complex involve the participation of an additional ionizable group with pK values of 7.30 and 6.30, respectively (Ikeda & Samejima (1981) J. Biochem. 90, 799-804), which was assigned as the alpha-amino group. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the intact and the alpha-NH2-modified enzymes was studied by the pH stat method at 25 degrees C, pH 8.2, and ionic strength 0.1 in the presence of 3 mM Ca2+. The Km value for the modified enzyme was found to be very similar to that for the intact enzyme: this was compatible with the results of the direct binding study on the monodispersed n-C12PC under the same conditions. However, the kcat value was about 43% of the value for the intact enzyme, suggesting that the alpha-keto group introduced by the chemical modification perturbed the network of hydrogen bonds in the active site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Kinetics of the hydrolysis of mixed micelles of dipalmitoyllecithin with triton X-100 catalyzed by a phospholipase A2 from the venom of Agkistrodon halys blomhoffii

The pH dependence of kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by the intact and the N-terminal alpha-NH2-modified phospholipases A2 (PLA2s) of Agkistrodon halys blomhoffii, was studied at 25 degrees C and ionic strength 0.1 in the presence of saturating amounts of Ca2+. The pH dependence of the kinetic parameters for the hydrolysis of monodispersed diC6PC, catalyzed by the modified enzyme, was also studied under the same conditions, and the data were compared with the previous results for the intact enzyme [Teshima, K. et al. (1986) J. Biochem. 100, 1655-1662]. The pK values of the catalytic group, His 48, and Tyr 52 were found to shift from 5.55 to 7.00 and from 10.50 to 11.50, respectively, on binding of the micellar substrates to the enzyme. On the other hand, no participation of these ionizable groups was observed for the binding of the monodispersed substrate. On the basis of the present finding and the X-ray crystallographic studies on bovine pancreatic PLA2 [Dijkstra, B.W. et. al. (1981) J. Mol. Biol. 147, 97-123] and on a PLA2 of Crotalus atrox venom [Brunie, S. et al. (1985) J. Biol. Chem. 260, 9742-9749], the hydrogen-bonding of Tyr 73, which is involved in the lipid-water interface recognition site, to His 48 and Tyr 52 in the active center was strongly suggested to be important for the hydrolysis of micellar substrates.     

pH dependence of the reaction rate of His 48 with p-bromophenacyl bromide and of the binding constant to Ca2+ of the monomeric forms of intact and alpha-NH2 modified phospholipases A2 from Trimeresurus flavoviridis

The phospholipase A2 of Trimeresurus flavoviridis was found to show monomer-dimer equilibria. Under conditions where the enzyme exists predominantly in the monomeric form, the chemical reaction rate of p-bromophenacyl bromide (BPB) with the catalytic group, His 48, was studied at 25 degrees C and ionic strength 0.2 by measuring the residual enzymic activity using a fluorescent substrate, 1,2-bis[4-(1-pyreno)butanoyl]-sn-glycero-3-phosphorylcholine (diPBPC). The pH-dependence curve of the reaction rate for the intact enzyme was practically the same as that for the modified enzyme, in which the N-terminal alpha-NH2 group had been selectively converted into an alpha-keto group. The pH-dependence curves were monophasic (sigmoidal) with a midpoint at pH 7.53, which corresponds to the pKa value of His 48. The pH dependences of the binding constants of Ca2+ to the intact and the alpha-NH2 modified enzymes were also studied at 25 degrees C and ionic strength 0.2 by measuring the changes in the tryptophyl fluorescence and/or aromatic CD spectra. The pH-dependence data for the modified enzyme were interpreted in terms of participation of Asp 49 (pKa 5.40) and His 48 (pKa 7.53), assuming that the protonation of Asp 49 competes with the Ca2+ binding. The pH-dependence data for the intact enzyme were similarly interpreted in terms of participation of the alpha-NH2 group (pKa 9.40) in addition to that of Asp 49 (pKa 5.40) and His 48 (pKa 7.53).(ABSTRACT TRUNCATED AT 250 WORDS)     

Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphorylcholine

Bindings of cobra venom phospholipases A2 to micelles of n-hexadecylphosphorylcholine were studied by the tryptophyl fluorescence method at 25 degrees C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of substrate (monomer) molecules, N = 10, 5 or 13 for N. naja atra apoenzyme and its Ca2+ complex, and N. naja kaouthia apoenzyme, respectively. The binding constant of the enzymes to the micelle, Kmic = 0.18-3.1 X 10(6) M-1, was 9-160 times greater than that to the monomeric substrate, Kmon = 2 X 10(4) M-1 (Teshima et al. (1981) J. Biochem. 89, 1163-1174). This was interpreted in terms of the presence of an additional substrate-binding site in the enzyme molecule. The binding constant of the enzyme-Ca2+ complex to the micelle was smaller than that for the apoenzyme over a wide range of pH. The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.4 to 5.53 and 7.55 to 7.95. The pH dependence curve for the Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of only a single ionizable group from 7.25 to 7.55. The former ionizable group was assigned as Asp 49, to which Ca2+ can coordinate, and the latter as His 48 in the active site on the basis of the reported pK values of these ionizable groups in the apoenzyme and Ca2+ complex (Teshima et al. (1981) J. Biochem. 89, 13-20 and Teshima et al. (1982) J. Biochem. 91, 1777-1788). No participation of the alpha-amino group with a pK value of 8.55 was observed.     

Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by a cobra (Naja naja atra) venom phospholipase A2

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by a cobra (Naja naja atra) venom phospholipase A2, was studied at 25 degrees C ionic strength 0.1 in the presence of 3-10 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micellar concentration (cmc), were analyzed according to the Michaelis-Menten equation. The Km value was practically independent of pH (between pH 6.75 and 10.30). This finding was consistent with the result of a direct binding study on monodispersed n-alkylphosphorylcholines (Teshima et al. (1981) J. Biochem. 89, 1163-1174). The hydrolysis of the substrate was competitively inhibited by the presence of monodispersed n-dodecylphosphorylcholine (n-C12PC). These results indicated that the substrate and n-C12PC compete for the same site on the enzyme molecule. The pH dependence curve of the kinetic parameter, kcat/Km, exhibited three transitions, below pH 8, between pH 8 and 9.5, and above pH 10. The analysis indicated the participation of three ionizable groups with pK values of 7.25, 8.50, and 10.4. The deprotonation of the first group and the protonation of the third group were found to be essential for the catalysis. The first group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Teshima et al. (1981) J. Biochem. 89, 13-20).(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of monodispersed and micellar substrates with a phospholipase A2 from Trimeresurus flavoviridis

Bindings of the phospholipase A2 from Trimeresurus flavoviridis to the monodispersed and micellar n-alkylphosphorylcholines (n-CnPC) were studied at 25 degrees C and ionic strength 0.2 by the aromatic CD and tryptophyl fluorescence methods, respectively. The bindings to micelles of the substrate analog were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 9-13. The binding constant to the micelle was about 40 times greater than it was to the monodispersed substrate. The binding constant to the micellar substrate analog increased on the binding of Ca2+ to the enzyme and decreased on modification of the N-terminal alpha-NH2 group, whereas the binding to the monodispersed substrate analog was independent of pH, of the Ca2+ binding, and of the chemical modification of the alpha-NH2 group. The kinetics of the hydrolyses of monodispersed and micellar dihexanoylphosphatidylcholines (diC6PC) were studied at 25 degrees C and ionic strength 0.2 by the pH-stat method in the presence of saturating amounts of Ca2+. The catalytic center activity, kappa cat, as well as the binding constant, 1/Km, for the micellar substrate, were found to be much greater than those for the monodispersed substrate. The binding constant, 1/Km, of the monodispersed substrate was independent of pH; this was in good agreement with that of the substrate analog described above. The pH-dependence curve of kappa cat for the monodispersed substrate exhibited two transitions, one below pH 6.5 and the other above pH 9.5.(ABSTRACT TRUNCATED AT 250 WORDS)     

pH dependence of the binding constant of a phospholipase A2 from Agkistrodon halys blomhoffii venom to micelles of n-hexadecylphosphorylcholine

The phospholipase A2 from the venom of A. halys blomhoffii was titrated with micellar n-hexadecylphosphorylcholine (an analog of lysolecithin) by following the tryptophyl fluorescence change at 25 degrees C and ionic strength 0.1. The data were analyzed by assuming that the micellar surface has multiple binding sites for the enzyme and that these sites are identical and mutually independent. The enzyme binding site was found to accommodate a constant number of the substrate (monomer) molecules, N = 10.0 and 6.7 for the apoenzyme and its Ca2+ complex, respectively. The binding constant of the enzyme to the substrate micelle was found to be enhanced by Ca2+ binding to the enzyme. The pH dependence of the binding constant of the apoenzyme to the micelle was well interpreted in terms of pK shifts of two ionizable groups from 5.16 to 5.67 and from 6.45 to 6.6. The pH-dependence curve for the enzyme-Ca2+ complex, which lacked the former transition, was interpreted in terms of the pK shift of a single ionizable group from 5.55 to 5.76. The former ionizable group was assigned as Asp 49, to which Ca2+ ion can coordinate, and the latter as His 48 in the active site. No participation of the alpha-amino group with a pK value of 7.30 was observed. The binding constant of the enzyme to the substrate micelle, Kmic = 0.45-2.3 X 10(6) M-1, was found to be far greater than that to the monomeric substrate, Kmon = 0.2-1.0 X 10(4) M-1. This was interpreted in terms of the presence of an additional weak substrate-binding site in the enzyme molecule.     

Kinetics of the hydrolysis of monodispersed dihexanoyllecithin catalyzed by the phospholipase A2 from Agkistrodon halys blomhoffii venom

The hydrolysis of 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by the phospholipase A2 from the venom of Agkistrodon halys blomhoffii, was studied at 25 degrees C and the ionic strength of 0.1 in the presence of 3-33.3 mM Ca2+, which can saturate the Ca2+-binding site of the enzyme. The initial velocity data, obtained at various concentrations of the substrate below the critical micelle concentration (cmc), were analyzed according to the Michaelis-Menten equation. The pH-dependence curve of the Km value exhibited only one transition below pH 8. The analytical results indicated that the pK value of 6.30 of an ionizable group changed to 6.54 on the binding of the monodispersed substrate. This ionizable group was assigned as the alpha-amino group on the basis of its pK value, which had been determined from the pH dependence of the binding constant of monodispersed n-dodecylphosphorylcholine (n-C12PC) (Ikeda and Samejima (1981) J. Biochem. 90, 799-804, and Haruki et al. (1986) J. Biochem. 99, 99-109). The pH-dependence curve of the kcat value exhibited two transitions, below pH 6.5 and above pH 9.5. The analytical results indicated the participation of two ionizable groups with pK values of 5.55 and 10.50. Deprotonation of the former and protonation of the latter group were found to be essential for the catalysis. The former ionizable group was assigned as His 48 in the active site on the basis of its pK value, which had been determined from the pH dependence of the binding constant of Ca2+ (Ikeda et al. (1981) J. Biochem. 90, 1125-1130).(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the hydrolysis of monodispersed dihexanoylphosphatidylcholine catalyzed by bovine pancreatic phospholipase A2: roles of Ca2+ binding and ionizations of amino acid residues in the active site.

Phospholipases A2 (PLA2s) are classified into two groups, I and II, according to differences in the polypeptide-chain length and intramolecular disulfide bondings. The hydrolysis of monodispersed 1,2-dihexanoyl-sn-glycero-3-phosphorylcholine (diC6PC), catalyzed by bovine pancreas PLA2 (Group I) was studied at 25 degrees C and ionic strength 0.2, and the initial velocity data were analyzed by means of the Michaelis-Menten equation. The Michaelis constant, Km, was found to be practically independent of Ca2+ concentration and also of pH value. The latter result indicates no participation of the ionizable groups in the active site in the substrate binding. The pH-dependence curve of the logarithm of the catalytic center activity, kcat, obtained in the presence of a practically saturating amount of Ca2+, showed three transitions ascribable to the participation of three ionizable groups with pK values of 5.00, 8.40, and 9.50. The respective groups were tentatively assigned to the catalytic group His 48, the N-terminal alpha-amino group, and invariant Tyr 52, which is located in close proximity to the imidazole ring of His 48. Deprotonation of His 48 and protonation of Tyr 52 were shown to be essential to the catalysis. The importance of the ionization state of the alpha-amino group was also indicated.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics of the hydrolysis of monodispersed and micellar phosphatidylcholines catalyzed by a phospholipase C from Bacillus cereus

The phosphatidylcholine-hydrolyzing phospholipase C, so-called "phospholipase C" (PLC), was isolated from the culture of Bacillus cereus strain IAM 1208. The amino-acid composition and partial N-terminal sequence of the purified enzyme were in good agreement with those expected from the nucleotide sequence for a PLC of strain ATCC 10987 [Johansen et al. (1988) Gene 65, 293-304]. The chain-length dependence of kinetic parameters for the PLC-catalyzed hydrolysis of monodispersed short-chain phosphatidylcholines (diCNPC, N = 3-6) was studied by a pH-stat assay method at 25 degrees C, pH 8.0, and ionic strength 0.2 in the presence of saturating amounts of Zn2+ (0.1 mM). The result was compared with those for snake venom phospholipases A2 [Teshima et al. (1989) J. Biochem. 106, 518-527]. It was found that the interaction of the PLC with the head group of the substrate molecule is very important for the binding. The pH dependences of kinetic parameters for the hydrolysis of monodispersed diC5PC and mixed micelles of diC16PC with Triton X-100 were also studied under the same conditions. An ionizable group, whose pK value is perturbed from 7.77 to 8.30 by substrate binding, was found to be essential to the catalysis. This group was tentatively assigned to His 14 on the basis of the results on X-ray crystallographic and chemical modification studies [Hough et al. (1989) Nature 338, 357-360 and Little (1977) Biochem. J. 167, 399-404].(ABSTRACT TRUNCATED AT 250 WORDS)     

Amino acid substitutions of the NH2-terminal Ala1 of porcine pancreatic phospholipase A2: a monolayer study

Previously it has been shown that the binding of porcine pancreatic phospholipase A2 to lipid-water interfaces is governed by the pK of the alpha-NH3+ group of the N-terminal alanine. Chemically modified phospholipases A2 in which the N-terminal Ala has been replaced by D-Ala or in which the polypeptide chain has been elongated with DL-Ala no longer display activity toward micellar substrate. The activity of DL-Ala-1-, [D-Ala1]-, and [Gly1]phospholipases A2 on substrate monolayers, which allow a continuous change in the packing density of the lipid molecule, was investigated. At pH 6 [Gly1]phospholipase A2 behaves like the native enzyme on lecithin monolayers. DL-Ala1- and [D-Ala1]phospholipases A2, although they are active in this system, showed a weaker lipid penetration capacity at this pH. Studies on the pH and Ca2+ ion dependency of the pre-steady-state kinetics and of the activity of these radiolabeled proteins showed that [D-Ala1]phospholipase A2 does not possess a second low-affinity site for Ca2+ ions in contrast to the native phospholipase A2. This second low-affinity Ca2+ binding site, which is also absent in [Gly1]phospholipase A2, is induced in the latter enzyme by the presence of lipid-water interfaces.     

Kinetics of the hydrolysis of micellar substrates catalyzed by snake venom phospholipases A2.

Effects of Ca2+ on the kinetic parameters for the hydrolysis of mixed micelles of 1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine (diC16PC) with Triton X-100, catalyzed by a cobra (Naja naja atra) (Group I) and a Habu (Trimeresurus flavoviridis) (Group II) PLA2s, were studied and compared with the results reported for other Group I and II enzymes. The substrate bindings to Group I enzymes were independent of the Ca2+ binding, whereas the substrate bindings to Group II enzymes were facilitated more than 10 times by the Ca2+ binding to the enzymes. The result for Group II enzymes, but not Group I enzymes, seemed compatible with the hypothesis for interpreting the catalytic mechanism that an intermediate complex should be stabilized by the coordination of the bound Ca2+ with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. The pH dependence of the kinetic parameters for the hydrolysis of the mixed micellar diC16PC, catalyzed by the cobra (N. naja atra) (Group I) and Habu (T. flavoviridis) (Group II) PLA2s, was also studied. The pK values of the catalytic group, His 48, and Tyr 52 for N. naja atra PLA2, shifted from 7.25 to 7.70 and from 10.30 to 10.85, respectively, and the corresponding values for T. flavoviridis PLA2 shifted from 5.80 to 6.95 and from 10.10 to 10.76, respectively, on binding of the micellar substrates to the enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Ca2+ in the substrate binding and catalytic functions of snake venom phospholipases A2

Phospholipases A2 are classified into two groups, I and II, according to differences in the polypeptide-chain length and the intramolecular-disulfide bondings. The effects of Ca2+ on the kinetic parameters for the hydrolysis of monodispersed and micellar phosphatidylcholines, catalyzed by a cobra (Naja naja atra) enzyme (Group I) and by mamushi (Agkistrodon halys blomhoffii) and habu (Trimeresurus flavoviridis) enzymes (Group II), were studied by the pH-statassay method at 25 degrees C, pH 8.0-8.2, and ionic strength 0.1-0.2. The results were compared with those reported for the other Group I and II enzymes. The Ca2+ binding was clearly shown to be essential for the catalysis of all the phospholipases A2. However, the substrate binding to Group I enzymes was found to be independent of the Ca2+ binding. On the other hand, the substrate binding to Group II enzymes was facilitated more than 10 times by the binding of Ca2+ to the enzymes. This was interpreted in terms of conformation changes of the peptide loop of residues 26 to 44 accompanying the Ca2+ binding. The latter result, but not the former, seems compatible with the hypothesis for interpreting the catalytic mechanism of phospholipases A2 that an intermediate complex should be stabilized by the coordination of the bound Ca2+ ion with the phosphoryl group and the carbonyl oxygen atom of the ester bond at the sn-2 position of the bound substrate molecule [Verheij et al. (1980) Biochemistry 19, 743-750 and (1981) Rev. Physiol. Biochem. Pharmacol. 91, 91-203]. According to the similarity in the primary and tertiary structures of the active sites of both types of enzymes [Renetseder et al. (1985) J. Biol. Chem. 260, 11627-11634], it is supposed that similar intermediate complexes may occur even for Group I enzymes, at least in the transition state of the productive complexes.     

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.     

The interaction of phospholipase A2 with micellar interfaces. The role of the N-terminal region.

The localization of the previously postulated interface recognition site (IRS) in porcine pancreatic phospholipase A2, required for a specific interaction between the enzyme and organized lipid-water interfaces, was investigated by ultraviolet difference spectroscopy, by measurements of the intrinsic fluorescence of the unique Trp residue, and by protection experiments against specific tryptic hydrolysis. Using the enzymically nondegradable substrate analogues: CnH(2n+1)(0-)OOCH2CH2N+(CH3)3-(H,OH), it is shown that the rather hydrophobic N-terminal sequence of the enzyme, viz., Ala-Leu-Trp-Gln-Phe-Arg, is directly involved in the interaction with the lipid-water interface. Besides hydrophobic probably also polar interactions contribute to the binding process. At neutral or acidic pH the presence of a salt bridge between the N-terminal alpha-NH3+ group and a negatively charged side chain stablizes the interface recognition site and allows the enzyme to penetrate micellar surfaces, even in the absence of metal ion. At alkaline pH, interaction of the enzyme with micellar interfaces requires the presence of Ca2+ (Ba2+) ions.     

Roles of asp126 and asp156 in the enzyme function of sphingomyelinase from Bacillus cereus.

To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/Km and kcat) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.     

Engineering the pH-dependence of kinetic parameters of maize glutathione S-transferase I by site-directed mutagenesis

The optimisation of enzymes for particular application or conditions remains an important target in all protein engineering endeavours. Here, we report a successful strategy for altering the pH-profile of kinetic parameters and to define in detail the molecular mechanism of maize glutathione S-transferase I (GST I). To accomplish this, selected residues from the glutathione binding site (His40, Ser11, Lys41, Asn49, Gln53 and Ser67) were mutated to Ala, and the pH-dependence of the catalytic parameters V(max), and V(max)/K(GSH)(m) of the mutated forms were analysed. The pH-dependence of V(max) for the wild-type enzyme exhibits two transitions in the acidic pH range with pK(a1) of 5.7 and pK(a2) of 6.6. Based on thermodynamic data, site-directed mutagenesis and UV deference spectroscopy, it was concluded that pK(a1) corresponds to GSH carboxylates, whereas the pK(a2) has a conformational origin of the protein. The pH-dependence of V(max)/K(GSH)(m) for the wild-type enzyme exhibits a single transition with pK(a) of 6.28 which was attributed to the thiol ionisation of bound GSH. These findings complement the conclusions about the catalytic mechanism deduced from the crystal structure of the enzyme and provide the basis for rationally designing engineered forms of GST I with valuable properties.     

Crosslinking by thiol disulfide interchange of 5,5'-dithiobis(2-nitrobenzoic acid)-treated light chain and heavy chain of rabbit skeletal myosin

Interchain disulfide crosslinks between the heavy-chain fragment in heavy meromyosin and myosin light chain 2, generated by 5,5'-dithiobis(2-nitrobenzoic acid (Nbs2), are formed under appropriate ionic conditions at neutral pH as revealed by liberation of the chromogenic 2-nitro-5-thiobenzoic acid. The presence of the original or of a slightly digested light chain 2 reduces the rate of the reaction of heavy meromyosin with Nbs2-modified light chain 2 by 32 - 39%, if Ca2+ is present. Dodecyl sulfate/polyacrylamide gel electrophoresis in absence of reducing agents shows that Nbs2-modified light chain 2 attaches to the heavy chain in the region of the 21-kDa fragment of heavy meromyosin, which contains the essential thiol groups and which has been located at the subfragment 1/subfragment 2 junction of myosin [Balint, M., Wolf, I., Tarcsafalvi, A., Gergely, J. and Sreter, F. A. (1978) Arch. Biochem. Biophys. 190, 793-799]. Modification of thiol-1 groups with iodoacetamide as well as crosslinking the thiol-1 and thiol-2 groups by the bifunctional reagent p-N,N'-phenylenedimaleimide prior to incubation with Nbs2-modified light chain 2 has no substantial effect on the crosslinking reaction. This indicates that other thiol groups are involved in the binding of Nbs2-modified light chain 2 to the heavy chain. An examination of K+, Ca2+, Mg2+ and actin-activated Mg2+ ATPase activities of heavy meromyosin that had been crosslinked with Nbs2-modified light chain 2 shows only a slight change in comparison with intact heavy meromyosin, indicating that crosslinking had not altered significantly the hydrolytic site. Crosslinking of Nbs2-modified light chain 2 to light-chain-2-deficient heavy meromyosin restored the original light-chain-2-dependent Ca2+ sensitivity of the tryptic fragmentation of heavy meromyosin, suggesting that crosslinking takes place at the proper binding site for light 2.     

Role of the N-terminus in the interaction of pancreatic phospholipase A2 with aggregated substrates. Properties and crystal structure of transaminated phospholipase A2

A free N-terminal alpha-NH3+ group is absolutely required for full catalytic activity of phospholipase A2 on aggregated substrates. To elucidate how this alpha-NH3+ group triggers catalytic activity, we specifically transaminated this group in various pancreatic phospholipases A2. Porcine, porcine iso-, equine, human, ovine, and bovine phospholipases A2 all loose catalytic activity on micellar substrates due to the inability of the transaminated proteins to bind to neutral micellar substrate analogues, as was found for the zymogens. Loss of activity is pseudo first order, the rate constants being different for the enzymes studied. The transaminated phospholipases A2 have an intact active site, as catalytic activities on monomeric substrates are comparable to those of the respective zymogens. The X-ray structure of transaminated bovine phospholipase A2 at 2.1-A resolution shows that the N-terminal region and the sequence 63-72 in this protein are more flexible than in the native enzyme. Also, in this respect, the transaminated enzyme very much resembles the zymogen structure. In good agreement with this, it was found by photochemically induced dynamic nuclear polarization 1H NMR that aromatic resonances of Trp-3 and Tyr-69 are affected by transamination. In addition, fluorescence spectroscopy of the unique Trp-3 in transaminated bovine phospholipase A2 revealed a red shift of the emission maximum indicative of a more polar environment of Trp-3 in the transaminated phospholipase A2 as compared to the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.