Comparative phosphorescence and optically detected magnetic resonance studies of fatty acid binding to serum albumin

The binding of free fatty acid to bovine serum albumin (BSA) and human serum albumin (HSA) was studied by phosphorescence and optical detection of triplet-state magnetic resonance spectroscopy in zero applied magnetic field. We have found that oleic acid perturbs the excited triplet state of Trp-134 but not that of Trp-212 in BSA. The assignment is made by comparing the BSA results with those obtained from oleic acid binding to HSA. The phosphorescence 0,0 band as well as the zero-field splittings of Trp-134 undergoes significant changes upon binding of oleic acid to BSA. Shifts of the 0,0-band wavelength and of the zero-field splittings point to large changes in the Trp-134 local environment which accompany the complex formation. The shifts are progressive until 3-4 mol of oleic acid is added. The spectroscopic changes may be attributed to Stark effects caused by a protein conformational change near Trp-134 in the BSA-oleate complex. Oleic acid binding has a minimal effect on the triplet-state properties of the single Trp-214 of HSA. The binding specificity with regard to chain length and unsaturation is reflected by the differences in the Trp environment when BSA forms complexes with various fatty acids.     

Investigation of phospholipase-lipid interactions by optical detection of triplet state magnetic resonance spectroscopy

We have investigated the binding of porcine pancreatic phospholipase A2 (PA2) to n-hexadecylphosphocholine (C16PN) micelles using optical detection of triplet state magnetic resonance (ODMR) spectroscopy. The zero field splittings (zfs) of the single Trp3 residue undergo significant changes upon binding of PA2 to C16PN micelles. Zfs titrations of PA2 vs C16PN indicate that the binding stoichiometry is C16PN:PA2 approximately 25. A reduction of the (E) parameter from 1.227 to 1.135 GHz is postulated to result from Stark effects caused by the binding of a polar group (possibly phosphocholine) near Trp3 in the PA2-C16PN micelle complex.     

Phosphorescence and optically detected magnetic resonance measurements of the 2'AMP and 2'GMP complexes of a mutant ribonuclease T1 (Y45W) in solution: correlation with X-ray crystal structures.

Phosphorescence and ODMR measurements have been made on ribonuclease T1 (RNase T1), the mutated enzyme RNase T1 (Y45W), and their complexes with 2'GMP and 2'AMP. It is not possible to observe the phosphorescence of Trp45 in RNase T1 (Y45W). Only that of the naturally occurring Trp59 is seen. The binding of 2'GMP to wild-type RNase T1 produces only a minor red shift in the phosphorescence and no change in the ODMR spectrum of Trp59. However, a new tryptophan 0,0-band is found 8.2 nm to the red of the Trp59 0,0-band in the 2'GMP complex of the mutated RNase T1 (Y45W). Wavelength-selected ODMR measurements reveal that the red-shifted emission induced by 2'GMP binding, assigned to Trp45, occurs from a residue with significantly different zero-field splittings than those of Trp59, a buried residue subject to local polar interactions. The phosphorescence red shift and the zero-field splitting parameters demonstrate that Trp45 is located in a polarizable environment in the 2'GMP complex. In contrast with 2'GMP, binding of 2'AMP to RNase T1 (Y45W) induces no observable phosphorescence emission from Trp45, but leads only to a minor red shift in the phosphorescence origin of Trp59 in both the mutated and wild-type enzyme. The lack of resolved phosphorescence emission from Trp45 in RNase T1 (Y45W) implies that the emission of this residue is quenched in the uncomplexed enzyme. We conclude that local conformational changes that occur upon binding 2'GMP remove quenching residues from the vicinity of Trp45, restoring its luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Changes in Ca2+ affinity upon activation of Agkistrodon piscivorus piscivorus phospholipase A2

Changes in the affinity of calcium for phospholipase A2 from Agkistrodon piscivorus piscivorus during activation of the enzyme on the surface of phosphatidylcholine vesicles have been investigated by site-directed mutagenesis and fluorescence spectroscopy. Changes in fluorescence that occur during lipid binding and subsequent activation have been ascribed to each of the three individual Trp residues in the protein. This was accomplished by generating a panel of mutant proteins, each of which lacks one or more Trp residues. Both Trp21, which is found in the interfacial binding region, and Trp119 show changes in fluorescence upon protein binding to small unilamellar zwitterionic vesicles or large unilamellar vesicles containing sufficient anionic lipid. Trp31, which is near the Ca2+ binding loop, exhibits little change in fluorescence upon lipid bilayer binding. A change in the fluorescence of the protein also occurs during activation of the enzyme. These changes arise from residue Trp31 as well as residues Trp21 and Trp119. The calcium dependence of the fluorescence change of Trp31 indicates that the affinity of the enzyme for calcium increases at least 3 orders of magnitude upon activation. These studies suggest either that a change in conformation of the enzyme occurs upon activation or that the increase in calcium affinity reflects formation of a ternary complex of calcium, enzyme, and substrate.     

Comparative triplet-state properties of the three tryptophan residues in bacteriophage T4 lysozyme and in the enzyme complex with methylmercury(II)

Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane.     

Optical detection of triplet-state magnetic resonance studies on individual tryptophan residues of serum albumin: correlation between phosphorescence and zero-field splittings

Cyanogen bromide cleavage of bovine serum albumin (BSA) yields two fragments, N (1-183) and C (184-582), containing 183 and 399 amino acid residues, respectively. Each in each fragment are characterized in this study by phosphorescence and optically detected magnetic resonance spectroscopy, and the results are compared with those of the intact albumin. Trp-134 in fragment N is located in a hydrophobic environment in the interior of the protein, as reflected by its red-shifted phosphorescence and characteristic zero-field splittings. The spectral properties of Trp-212 in fragment C suggest its location in a partially buried, inhomogeneous environment. They show great similarity to those of human serum albumin, which contains a single Trp at position 214. The Trp phosphorescence 0,0-bands of fragments C and N are fitted with Gaussian functions by computer, and their relative contributions to the phosphoresence 0,0-band of BSA are adjusted to fit the observed BSA 0,0-band. The wavelength dependence of the [D[-[E[ transition frequencies of fragments N and C is then weighted by their 0,0-band intensity, taking into account differences in spin alignment, and summed to predict the peak frequency of the [D[-[E[ band profile as a function of phosphorescence wavelength for the intact BSA. Good agreement between predicted and observed behavior of [D[-[E[ vs. wavelength for the intact protein provides strong evidence for the additivity of the phosphorescence and ODMR spectra of the individual Trp sites in BSA. We find that Trp-134 and Trp-212 have wavelength-independent and wavelength-dependent zero-field splittings, respectively.     

Photo-CIDNP 1H-NMR studies of bovine pancreatic phospholipase A2 and its zymogen

Bovine pancreatic phospholipase A2 and its zymogen were studied by laser photo-CIDNP 1H-NMR. Resonances of Trp3 and Tyr69 protons of the two proteins were assigned. By varying the delay between a short light pulse and the observation pulse, time dependencies of the CIDNP signals were obtained from which effective T1 values could be derived. The photo-CIDNP chemical shifts, intensities and relaxation data pointed to environmental differences for the Tyr69 residues in the two proteins, while only small differences were noted for the Trp3 residues. The more buried position of Tyr69 in the enzyme relative to the zymogen was related to the ability of the enzyme to bind to micellar aggregates, to which the zymogen is unable to bind.     

Unusual kinetic behavior of porcine pancreatic (pro)phospholipase A2 on negatively charged substrates at submicellar concentrations

The negatively charged detergents S-n-alka-noylthioglycol sulfates (C8, C9, and C10) are substrates for porcine pancreatic phospholipase A2 and its zymogen. At pH 6.0 and detergent concentrations up to 0.08 X critical micelle concentration (cmc), the activities of active enzyme and zymogen are similar and very low. From 0.08 X cmc to 0.12 X cmc a tremendous increase in activity is observed for phospholipase A2, but not for the zymogen. Concomitant with this increase in activity there is a sharp rise in molecular weight of the substrate-enzyme complex, from 15 000 to 95 000, and in detergent to protein molar ratio of 1:1 to about 7:1. This indicates both substrate and enzyme aggregation. Most probably a lipid-water interface is formed inside the aggregated protein particle by which the enzyme is activated. Although the zymogen also forms high molecular weight complexes with similar molar ratios, no activation is observed probably because of distortion of its lipid binding domain.     

Evidence that the zymogen of phospholipase A2 binds to a negatively charged lipid-water interface

Evidence is presented that the zymogen of porcine pancreatic phospholipase A2 (prophospholipase A2) interacts with a lipid-water interface provided that the interface has a net negative surface charge. Fluorescence spectroscopy and non-equilibrium gel filtration indicate that binding of prophospholipase A2 (proPLA) to mixed detergent micelles is dependent on the presence of an anionic detergent. Prophospholipase binding is accompanied by a change in the environment of the single tryptophan residue qualitatively similar to that observed when the active enzyme, phospholipase A2 (PLA), binds to micelles. In addition, the rate of tryptic activation of prophospholipase is significantly reduced in the presence of negatively-charged mixed micelles, whereas no change in rate occurs when neutral micelles are present. These observations suggest that the lack of catalytic activity of the zymogen toward organized substrates carrying a negative surface charge cannot be explained by a failure to bind at the lipid-water interface.     

Orientations of the tryptophan 9 and 11 side chains of the gramicidin channel based on deuterium nuclear magnetic resonance spectroscopy.

Deuterium nuclear magnetic resonance spectroscopy was used to investigate the orientations of the indole rings of Trp9 and Trp11 in specific indole-d5-labeled samples of gramicidin A incorporated into dimyristoyl phosphatidylcholine bilayers in the beta 6.3 channel conformation. The magnitudes and signs of the deuterium quadrupolar splittings were fit to the rings and assigned to specific ring bonds, using a full rotation search of the chi 1 and chi 2 angles of each Trp and a least-squares method. Unique assignments were obtained. The data and assignments are in close agreement with four sets of (chi 1, chi 2) angles for each Trp in which the indole N-H is oriented toward the membrane's exterior surface. (Four additional sets of (chi 1, chi 2) angles with the N-H's pointing toward the membrane interior are inconsistent with previous observations.) One of the sets of (chi 1, chi 2) angles for each Trp is consistent with the corresponding Trp orientation found by Arsen'ev et al. (1986. Biol. Membr. 3:1077-1104) for gramicidin in sodium dodecyl sulfate micelles. Together, the 1H and 2H nuclear magnetic resonance methods suggest that the Trp9 and Trp11 side chain orientations could be very similar in dimyristoyl phosphatidylcholine membranes and in sodium dodecyl sulfate micelles. The data for Trp11 could be fit using a static quadrupolar coupling constant of 180 kHz under the assumption that the ring is essentially immobile. By contrast, Trp9 could be fit only under the assumption that the quadrupolar splittings for ring 9 are reduced by approximately 14% due to motional averaging. Such a difference in motional averaging between rings 11 and 9 is also consistent with the 15N data of Hu et al. (1993. Biochemistry. 32:7035-7047).     

Molecular ordering of interfacially localized tryptophan analogs in ester- and ether-lipid bilayers studied by 2H-NMR.

Perdeuterated indole-d6 and N-methylated indole-d6 were solubilized in lamellar liquid crystalline phases composed of either 1,2-diacyl-glycero-3-phosphocholine (14:0)/water or 1,2-dialkyl-glycero-3-phosphocholine(14:0/water. The molecular ordering of the tryptophan analogs was determined from deuteron quadrupole splittings observed in 2H-NMR spectra on macroscopically aligned lipid bilayers. NMR spectra were recorded with the bilayers oriented perpendicular to or parallel with the external magnetic field, and the values of the splittings differed by a factor of 2 between these distinct orientations, indicating fast rotational motion of the molecules about an axis parallel to the bilayer normal. In all cases the splittings were found to decrease with increasing temperature. Relatively large splittings were observed in all systems, demonstrating that the tryptophans partition into a highly anisotropic environment. Solubilization most likely occurs at the lipid/water interface, as indicated by 1H-NMR chemical shift studies. The 2H-NMR spectra obtained for each analog were found to be rather similar in ester and ether lipids, but with smaller splittings in the ether lipid under similar conditions. The difference was slightly less for the indole molecule. Furthermore, in both lipid systems the positions of the splittings from indole were different from those of N-methyl indole. The results suggest that 1) the tryptophan analogs are solubilized in the interfacial region of the lipid bilayer, 2) the behavior may be modulated by hydrogen bonding in the case of indole, and 3) hydrogen bonding with the lipid carbonyl groups is not likely to play a major role in the solubilization of single indole molecules in the ester lipid bilayer interface.     

Optically detected magnetic resonance study of the interaction of an arsenic(III) derivative of cacodylic acid with EcoRI methyl transferase

The interaction of the enzyme Escherichia coli RI methyl transferase (methylase) with an arsenic(III) derivative of cacodylic acid has been investigated by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy in zero applied magnetic field. The reactive derivative (CH3)2AsSR is formed by the reduction of cacodylate by a thiol. The As(III) derivative binds to the enzyme by mercaptide exchange with a cysteine (Cys) residue located close to a tryptophan (Trp) site. The arsenical binding selectively induces an external heavy-atom effect, perturbing the nearby Trp residue in the enzyme. Zero-field splittings (ZFS) and total decay rate constants of the individual triplet-state sublevels of the Trp residue in the presence and absence of perturbation by As(III) have been determined. The perturbed Trp shows a large reduction in the overall decay lifetime compared with unperturbed Trp residue, exhibiting a high selectively for the Tx sublevel. This selectivity suggests that the As atom lies in the xz plane of the principal magnetic axis system of Trp, but not directly along the z (out-of-plane) axis. The accessibility of this enzyme binding site to the arsenical is decreased upon forming a ternary complex of methylase with sinefungin and a DNA oligomer, d[GCGAA(BrU)(BrU)CGC], containing two 5-bromouracil (BrU) bases in place of thymine within the hexadeoxynucleotide recognition sequence. This result indicates that the arsenical binding site in methylase which produces the Trp heavy-atom effect is protected from this ligand by ternary complex formation or the enzyme undergoes a conformation change, removing the Cys from the Trp site. This protection is also observed in fluorescence quenching experiments.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipoproteins of dimyristoylphosphatidylcholine

The structure of the exchangeable apolipoprotein, apolipophorin-III from Locusta migratoria, apoLp-III, is described as a bundle of five amphipathic alpha-helices. To study the interaction of each of the helices of apoLp-III with a lipid surface, we designed five single-Trp mutants, each containing a Trp residue in a different alpha-helix. The Trp residues were located in the nonpolar domains of the amphipathic alpha-helices. The kinetics of the spontaneous interaction of the mutants with dimyristoylphosphatidylcholine (DMPC) indicated that all mutants behaved as typical exchangeable apolipoproteins. Circular dichroism in the far-UV indicated that all proteins have a high and similar helical content in the lipid-bound state. The interaction of the Trp residues with the lipid surface was investigated in recombinant lipoprotein particles made with DMPC. The properties of the Trp residues were investigated by fluorescence spectroscopy. These studies showed major changes in the spectroscopic properties of the Trp residues upon binding to lipid. These changes are observed with all single-Trp mutants, indicating that a major conformational change, which affects the properties of all helices, takes place upon binding to lipid. The position of the fluorescence maximum, the quenching efficiency of acrylamide as determined by steady-state and time-resolved fluorescence, and the fluorescence lifetimes of the single-Trp mutants suggest that helices 1, 4, and 5 interact with the nonpolar domains of the lipid. The properties of the Trp in helices 2 and 3 suggest that these helices adopt a different binding configuration than helices 1, 4, and 5. Helices 2 and 3 appear to be interacting with the polar headgroups of the phospholipids or constitute a different domain that does not interact with the lipid surface.     

Specific transformations at the N-terminal region of phospholipase A2.

Treatment of porcine pancreatic prophospholipase A2 with methyl acetimidate converted all lysine residues into epsilon-acetimidolysine residues. Enzymatically active epsilon-amidinated phospholipase A2 (AMPA) was obtained from the epsilon-amidinated zymogen by limited tryptic proteolysis cleaving the Arg7-Ala8 bond. AMPA was used to prepare des-Ala8-, des-(Ala8,Leu9)- and des-(ALa8),Leu9,Trp10)-AMP by successive Edman degradations, and des-(A la 8-Arg13)-AMPA by selective splitting of the Arg13-Ser14 bond by trypsin. Structural analogues of AMPA with different N-terminal amino acid residues, viz., D-Ala, beta-Ala, and Gly, have been prepared by reacting des-Ala8-AMPA with the corresponding N-t-Boc-N-hydroxysuccinimide esters of these amino acids. Similarly, the only Trp10 residue has been substituted for Phe by coupling of des-(Ala8-,Leu9,Trp10)-AMPA with N-t-Boc-L-Ala-L-Leu-L-Phe-N-hydroxysuccinimide ester. The feasibility of these substitutions has been proven unambiguously by the retroconversion of des-Ala8-AMPA and of [Ala7]AMPA into AMPA having identical enzymatic activity as the starting AMPA. The single Trp10 residue in native phospholipase A2 and its zymogen was specifically sulfenylated using 0-nitrophenyl-sulfenyl chloride. The homogenous proteins were kinetically analyzed using short-chain lecithins in the monomeric and micellar region. All modified AMPA analogues, except those in which two or more of the N-terminal amino acid residues are removed, show enzymatic activities toward monermic substrate comparable to that of AMPA, indicating that the active site region is still intact. Only [Gly8]-, [beta-Ala8]-, and [Ala8,Leu9,Phe10]AMPA exhibit a dramatic increase in enzymatic activity similar to that of AMPA upon passing the critical micellar concentration (cmc) of the substrate. From these results it can be concluded that the N-terminal region of the enzyme requires a very precise architecture in order to interact with lipid-water interfaces and consequently to display its full enzymatic activity.     

Energy transfer in complexes of E. coli single-stranded DNA-binding protein with single-stranded poly-(2-thiouridylic acid)

The complexes of point-mutated Escherichia coli single-stranded DNA-binding protein (Eco SSB) with poly-(2-thiouridylic acid) (poly S2U) have been studied by optical detection of magnetic resonance spectroscopy (ODMR). Previous work has determined that two of four tryptophan (Trp) residues in Eco SSB undergo stacking interactions with nucleic acid bases. Selective photoexcitation of S2U bases was performed and subsequent triplet----triplet energy transfer from S2U to nearby Trp residues in the protein took place. The zero-field splitting (ZFS) parameters and sublevel kinetics were determined for each Trp residue sensitized by S2U. The sublevel lifetimes of the two sensitized residues are similar to those of normal Trp. The ZFS parameters, on the other hand, show a dramatic reduction relative to those of the uncomplexed protein, implying a more polarizable environment for the sensitized Trp residues and/or charge transfer interactions with the S2U bases.     

Structural basis of phospholipase A2 inhibition for the synthesis of prostaglandins by the plant alkaloid aristolochic acid from a 1.7 A crystal structure

This is the first structural observation of a plant product showing high affinity for phospholipase A(2) and regulating the synthesis of arachidonic acid, an intermediate in the production of prostaglandins. The crystal structure of a complex formed between Vipera russelli phospholipase A(2) and a plant alkaloid aristolochic acid has been determined and refined to 1.7 A resolution. The structure contains two crystallographically independent molecules of phospholipase A(2) in the form of an asymmetric dimer with one molecule of aristolochic acid bound to one of them specifically. The most significant differences introduced by asymmetric molecular association in the structures of two molecules pertain to the conformations of their calcium binding loops, beta-wings, and the C-terminal regions. These differences are associated with a unique conformational behavior of Trp(31). Trp(31) is located at the entrance of the characteristic hydrophobic channel which works as a passage to the active site residues in the enzyme. In the case of molecule A, Trp(31) is found at the interface of two molecules and it forms a number of hydrophobic interactions with the residues of molecule B. Consequently, it is pulled outwardly, leaving the mouth of the hydrophobic channel wide open. On the other hand, Trp(31) in molecule B is exposed to the surface and moves inwardly due to the polar environment on the molecular surface, thus narrowing the opening of the hydrophobic channel. As a result, the aristolochic acid is bound to molecule A only while the binding site of molecule B is empty. It is noteworthy that the most critical interactions in the binding of aristolochic acid are provided by its OH group which forms two hydrogen bonds, one each with His(48) and Asp(49).     

Effect of Phospholipase Digestion and Lysophosphatidylcholine on Dopamine Receptor Binding

[3H]Spiperone specific binding by microsomal membranes isolated from sheep caudate nucleus is decreased by trypsin and phospholipase A2 (Vipera russeli), but is insensitive to neuraminidase. The inhibitory effect of phospholipase A2 is correlated with phospholipid hydrolysis. After 15 min of phospholipase (5 micrograms/mg protein) treatment, a maximal effect is observed; the maximal lipid hydrolysis is about 56% and produces 82% reduction in [3H]spiperone binding. Equilibrium binding studies in nontreated and treated membranes showed a reduction in Bmax from a value of 388 +/- 9.2 fmol/mg protein before phospholipase treatment to a value of 52 +/- 7.8 fmol/mg protein after treatment, but no change in affinity (KD = 0.24 +/- 0.042 nM) was observed. Albumin washing of treated membranes removes 47% of lysophosphatidylcholine produced by phospholipid hydrolysis without recovering [3H]spiperone binding activity. However, the presence of 2.5% albumin during phospholipase A2 action (1.5 micrograms/mg protein) prevents the inhibitory effect of phospholipase on [3H]spiperone binding to the membranes, although 28% of the total membrane phospholipid is hydrolysed. Lysophosphatidylcholine, a product of phospholipid hydrolysis, mimics the phospholipase A2 effect on receptor activity, but the [3H]spiperone binding inhibition can be reversed by washing with 2.5% defatted serum albumin. Addition of microsomal lipids to microsomal membranes pretreated with phospholipase does not restore [3H]spiperone stereospecific binding. It is concluded that the phospholipase-mediated inhibition of [3H]spiperone binding activity results not only from hydrolysis of membrane phospholipids, but also from an alteration of the lipid environment by the end products of phospholipid hydrolysis.     

Investigation of the complexes of EcoRI endonuclease with decanucleotides containing canonical and modified recognition sequences using fluorescence and optical detection of magnetic resonance spectroscopy

The binding of EcoRI endonuclease to the oligonucleotides d(GCGAATTCGC) and d(GCGAA) (5BrdU) (5BrdU) d(CGC) has been investigated to determine whether stacking interactions occur between tryptophan residues and the DNA bases. Fluorescence binding isotherms show that the decamer containing the canonical and that containing the modified recognition sequence bind with comparable affinity. Optically detected magnetic resonance spectra show limited perturbations of the Trp zero-field splitting parameters, which are assigned to electrical field effects. No evidence for Trp stacking interactions has been found.     

Desolvation map of the i-face of phospholipase A2

The changes in the microenvironment of the Trp-3 on the i-face of pig pancreatic IB phospholipase A2 (PLA2) provide a measure of the tight contact (Ramirez and Jain, Protein Sci. 9, 229-239, 1991) with the substrate interface during the processive interfacial turnover. Spectral changes from the single Trp-substituent at position 1, 2, 6, 10, 19, 20, 31, 53, 56 or 87 on the surface of W3F PLA2 are used to probe the Trp-environment. Based on our current understanding only the residue 87 is away from i-face, therefore all other mutants are well suited to report modest differences along the i-face. All Trp-mutants bind tightly to anionic vesicles. Only those with Trp at 1, 2 or 3 near the rim of the active site on the i-face cause significant perturbation of the catalytic functions. Most other Trp-mutants showed < 3-fold change in the interfacial processive turnover rate and the competitive inhibition by MJ33. Binding of calcium to the enzyme in the aqueous phase had modest effect on the Trp-emission intensity. However, on the binding of the enzyme to the interface the fluorescence change is large, and the rate of oxidation of the Trp-substituent with N-bromosuccinimide depends on the location of the Trp-substituent. These results show that the solvation environment of the Trp-substituents on the i-face is shielded in the enzyme bound to the interface. Additional changes are noticeable if the active site of the bound enzyme is also occupied, however, the catalytically inert zymogen of PLA2 (proPLA2) does not show such changes. Significance of these results in relation to the changes in the solvent accessibility and desolvation of the i-face of PLA2 at the interface is discussed.     

Calcium ion binding to pancreatic phospholipase A2 and its zymogen: a 43Ca NMR study

Calcium ion binding to phospholipase A2 and its zymogen has been studied by 43Ca NMR. The temperature dependence of the band shape of the calcium-43 NMR signal has been used to calculate the calcium ion exchange rate. The on-rate was calculated to be 5 X 10(6) M-1 s-1, which is 2 orders of magnitude less than the diffusion limit of the hydrated Ca2+ ion in water. The 43Ca quadrupole coupling constant for calcium ions bound to phospholipase, chi = 1.4 MHz, is significantly larger than those found for EF-hand proteins, indicating a less symmetric site. For prophospholipase A2, we found chi = 0.8 MHz, indicating a calcium binding site, which is somewhat more symmetric than the EF-hand sites. The dependence of the 43Ca NMR band shape on the calcium ion concentration showed that there are two cation binding sites on the phospholipase A2 molecule: K1 = 4 X 10(3) M-1 and K2 = 20 M-1. The strong site was found to be affected by a pKa = 6.5 and the weak site by pKa = 4.5.