Biochemistry 1994, 33, 4509-4516
4509
Functional Significance of the Conformational Dynamics of the N-Terminal Segment of Secreted Phospholipase A2 at the Interface? Badri P. Maliwal,'.*.s Bao-Zhu Y U , ~ Henryk Szmacinski,* Thomas Squier,* Jan van Binsbergen,l Arend J. Slotboom,l and Mahendra K. Jain'vs Center for Fluorescence Spectroscopy, Department of Biological Chemistry, University of Maryland Medical School, Baltimore, Maryland 21 201, Department of Chemistry, University of Delaware, Newark, Delaware 1971 6, and Department of Biochemistry, Rijksuniversiteit, Padualaan 8, Utrecht. The Netherlands Received August 17, 1993; Revised Manuscript Received December IO, 1993'
ABSTRACT: The kinetic and fluorescence properties of several pig pancreatic phospholipase A2 (PLA2) with substitutions and deletions in the N-terminal region and of tyrosines 52 and 73 are characterized. The substitutions Ala-1-D-Ala or -Gly, Trp-3-Phe, Gln-4-Nle, Arg-6-Glu, Tyr-52-Phe, and Tyr-73-Phe had at the most only a modest effect on the interfacial catalytic activity on the anionic interface to which they bind with high affinity. The observed rate of hydrolysis in the scooting mode by deletion mutants lacking one or more successive residues from the N-terminal region was lower by 50-95%. Detailed kinetic analysis of the deletion mutant lacking Ala-1 (des-1-AMPA) showed that the 50% decrease in the rate is due to a 5-fold increase in the interfacial Michaelis-Menten parameter, KM*,without a significant change in kat, These results and direct measurements show that the primary effect of Ala-1 deletion is to lower the affinity for the active site directed ligands. Although the affinity of these mutants for anionic interface remains the same as for the wild type, the affinity for zwitterionic neutral diluents is considerably lower. Significant differences in the fluorescence quantum yields and the heterogeneity in the frequency-domain fluorescence intensity decays of these enzymes suggest that both in solution and at the interface the N-terminal region is an ensemble of conformations rather than a discrete state. Additional results suggest that the interfacial microenvironment of Trp-3 in des-1-AMPA is more polar and Trp-3 is more accessible to quenching by acrylamide. This indicates incomplete desolvation of the microinterface in case of des- 1-AMPA. The frequency-domain anisotropy decays of Trp-3 indicate that the N-terminal region of free PLA2 is somewhat rigid and becomes flexible as Ala-1 and Leu-2 are deleted. On the other hand, on DTPM vesicles, the N-terminal region is essentially rigid in PLA2 and des-1-AMPA, and it is only modestly flexible in bound des- 1,2-AMPA. These observations demonstrate a ligand-induced rigidification of the N-terminal region of the E*I form of PLA2, and it is surprising that such a rigidification is observed in the absence of Ala-1. Taken together these results show that Ala-1 and the associated hydrogen-bond network affect the binding of an active site directed ligand via its role in desolvation of the interface but without a significant effect on the chemical step of the catalytic turnover cycle.
Interfacial catalytic behavior of phospholipase A2 (PLA2) is adequately described by the reaction sequence:
where the binding of the enzyme from the aqueous (E) to the interface (E*) precedes the catalytic turnover, suggesting a separation of the steps of catalytic turnover cycle (Verheij et al., 1981; Volwerk & de Haas, 1982). A support for this dissection is provided by the fact that the E, E*, and E*L forms of the enzyme have distinct spectroscopic signatures (Jain & Maliwal, 1993). A functional separation of these two events implies not only an increased residence time and enhanced catalytic processivity of the enzyme at the interface This work was supported by Grant GM29703 (M.K.J.) from the National Institutes of Health. We thank Professor Joseph Lakowicz for use of the facilities of the Center for Fluorescence Spectroscopy at the University of Maryland supported by Grants RR08 119 from theNationa1 Institutes of Health and Dir-8710401 from the National Science Foundation. University of Maryland Medical School. 8 University of Delaware. 1 Rijksuniversiteit. Abstract published in Advance ACS Abstracts, March 15, 1994. @
0006-2960/94/0433-4509$04.50/0
(Jain et al., 1986a) but it also provides a functional basis for understanding interfacial activation (Jain et al., 1993). Such analysis has provided a basis for deconvolutionof the interfacial rate and equilibrium parameters (Berg et al., 1991). The importance of obtaining insights into the structural basis for the intrinsic functional differences between the E and E* forms Abbreviations: The kinetic and equilibrium constants are defined conventionally except for the fact that the parameters marked with an asterisk relate to the equilibria at the interface. For example, KL and KL*values are the equilibrium dissociation constants for the EL and E*L complexes, where L is a substrate, product or inhibitor;&(50) is the mole fraction required for 50% inhibition; vo, is the measured initial rate at mole fraction = 1 of the substrate at the interface; kat is the calculated rate at the saturating substrate mole fraction based on the value of the interfacial Michaelis-Menten constant, KM*. Kd, dissociation constant for E* to E dissociation constant for E*Ito EI; AMPA, c-amidinated PLA2 from pig pancreas; deoxy-LPC, 1-hexadecylpropanediol-3-phosphocholine;des-1-AMPA, AMPA without the alanine at the N-terminus; des-1,2-AMPA, AMPA without Ala-1 and Leu-2 at the N-terminus; des-1,6-AMPA, AMPA without residues 1-6 at the N-terminus; des1,8-AMPA, AMPA without residues 1-8 at the N-terminus; DMPM, dimyristoylglycero-sn-3-phosphomethanol; DTPM, ditetradecylglycerosn-3-phosphomethanol; E, E*, andE*L, theenzymein the aqueousphase, the enzyme at the interface, and the enzyme-ligand complex at the interface; MJ33, l-hexadecyl-3-(trifluoroethyl)-rac-glycero-2-phosphomethanol; M 572, 1-octyl-3-(trifluoroethyl)-rac-glycero-2-phosphomethanol; PLA2, native phospholipase A2 from pig pancreas unless stated otherwise.
0 1994 American Chemical Society
4510 Biochemistry, Vol. 33, No. 15, I994 is obvious, and such attempts may be viewed in terms of two different hypotheses elaborated below. According to the "enzyme conformation" theory apparently higher catalytic turnover by E* is the result of a discrete conformational transition that occurs on the binding of the enzyme to the interface (Volwerk & de Haas, 1982). Putatively, the H-bond network involving Ala- 1 along with the N-terminal helix stabilizes the E* form (Dijkstra et al., 1981, 1983). In the X-ray crystal structures of bovine and porcine PLA2 and their complexeswith inhibitors, the a-NHs+ group of Ala- 1 is hydrogen bonded to nearly invariant Gln-4 and Glu(Asn)-7 1 and is linked by a structural water molecule to invariant residues Tyr-52, Tyr-73, and Asp-99, the last residue being part of the "catalytic triad". Hydrogen bonds with the carbonyl from the backbone of residues Pro-68 and Glu-71 are also implicated in this network. In addition, residues Leu-2, Phe-5, and Ile-9 form a part of the wall of the active-sitecavity (Scott et al., 1990; Thunnissen et al., 1990). Multidimensional NMRstudies of E and E*I complex indicate that the hydrogen-bond network is fully formed only when an active site directed ligand binds to the enzyme at the interface (Dekker et al., 1991a,b; Peters et al., 1992). Thus, according to this hypothesis, the interfacial activation of the enzyme is brought about by the formation of this hydrogen-bond network (Volwerk & de Haas, 1982). Several observations raise doubt about a crucial role for such a discrete two-state conformational transition and the H-bond network (a) The remarkable similarity in the catalytic surfaces among evolutionarily divergent PLA2 and between E and E1 complexes in the crystal structures suggests that the optimal active-site architecture is already present in the free enzyme (Scott et al., 1990; Thunnissen et al., 1990). (b) The affinity of PLA2 for the cofactor calcium is essentially the same for the E and E* forms (Jain et al., 1991a). (c) The role of hydrogen bonds involving tyrosines at 52 and 73 in the interfacial catalysis is virtually ruled out by normal enzyme activity observed with corresponding phenylalanine mutants of bovine (Dupureur et al., 1992a) and porcine (Kuipers et al., 1990) PLA2. On the basis of such considerations and dynamic fluorescence results, it has been suggested that the N-terminal segment of the E form of PLAZ exists as an ensemble of conformations (Jain & Maliwal, 1993). Upon binding to an interface (E to E*), the N-terminal segment of PLA2 becomes more rigid, representing a narrower ensemble of perhaps the optimally active conformation. Moreover, desolvation of the interface associated with the formation of E*L also plays a significant role in the enhanced interfacial activity (Jain & Vaz, 1987; Ramirez & Jain, 1991) due to an increased affinity of the bound enzyme for the substrate (Jain et al., 1993). In this paper we report studies designed to evaluate the role of the N-terminal region. The kinetic and equilibrium binding properties of several mutants of pig pancreatic PLA2 with substitution and deletion in the N-terminal region were determined. Parallel fluorescence studies were also carried out to characterize the conformational dynamics of the N-terminal region and thus provide a structural basis for the observed functional changes. These results show that the H-bonded network associated with Ala-1 is not necessary for the chemical step of the catalytic cycle, but it plays an important role in the binding of active site directed ligands to the enzyme at the interface. Although the affinity of the enzyme for the anionic interface is not noticeably altered by these mutations, the affinity for binding to zwitterionic interfaces does decrease significantly. In fact, this dissection
Maliwal et al. of the three steps provides a rationale for the loss of catalytic activity of these mutants on zwitterionic interfaces (Slotboom & de Haas, 1975; Van Scharrenburg et al., 1984a,b).
MATERIALS AND METHODS All reagents, proteins, and phospholipids used here were of better than 99% purity as checked by thin-layer chromatography or HPLC in appropriate solvent systems. Methods for the preparation of lipids (Jain et al., 1986a), neutral diluents (Jain et al., 1991a,b),competitiveinhibitors (Jain et al., 199IC), native, alkylated, amidinated, and semisynthetic PLA2 (Van Scharrenburg et al., 1984a,b) have been described. Tyr-52Phe, and Tyr-73-Phe mutants of the porcine PLAZ were kindly provided by Professor Verheij. The general protocols for monitoring the steady-state fluorescence and acrylamide quenching (Jain et al., 1986b; Jain & Maliwal, 1985, 1993), for obtaining the steady-state rate of hydrolysis, uo,expressed as turnover number, for interfacial catalysis in the scooting mode (Jain et al., 1991a,b), and for determining the rate and equilibrium parameters and the underlying analytical relations (Berg et al., 1991, Jain et al., 1991a, 1993; Yu et al., 1993) have been established. Uncertainties in the experimental values are less than 10% and the standard deviations for the derived parameters were less than 30%. All spectroscopic measurements were made in 10 mM Tris, 0.5 mM CaCl2 at pH 8.0 and 24 "C.Unless stated otherwise, for the steady-state fluorescence measurements the excitation wavelength was 292 nm and the protein concentration was between 3 and 10 pM with no noticeable inner filter effects. Steady-state fluorescence spectra were obtained on an SLM 4000 or 4800s spectrophotometer. The ultraviolet absorption spectra were measured on a HP8452 spectrophotometer equipped with a diode array detector, and the protein concentration was between 20 and 35 pM. The dynamic fluorescence measurements were performed on a 10-GHz frequency-domain harmonic content fluorometer as described (Laczko et al., 1990). The harmonic content of a rhodamine6G dye laser, synchronouslypumped with a modelocked argon ion laser, cavity dumped at 3.75 MHz, and frequency-doubled to 295 nm was used as an excitation source. The emission was observed through Corning 0-54 and 7-54 filters which provided a broad emission window. The protein concentration was between 20 and 50 pM, and a 4-mm pathlength cuvette was used. The frequency responses of the emission intensity decays were analyzed by nonlinear leastsquares fitting routines as a sum of exponentials (Lakowicz et al., 1984):
where a and T are amplitudes and the associated lifetime components. The average life time ( 7 ) was computed as
All related intensity decays were globally analyzed for three common and one variable exponential decay components. Such analysis gave same xR2valuesas separate, unrestrained analysis of each file but significantly reduced the uncertainty in the resolved parameters. The anisotropy decays [r(t)] were analyzed as a sum of exponentials as follows (Weber, 1977; Maliwal & Lakowicz, 1986):
Biochemistry, Vol. 33, No. 15, 1994 45 11
Dynamics of the N-Terminal Segment Table 1: Catalytic Turnover Numbers (vo) and Relative Quantum Yields (RQY) of PLA2, Semisynthetic AMPA, and the Tyr Mutants in the Scooting Mode Assay with DMPM mutant PLAZ pro-PLA2 AMPA Ala-1-Gly-AMPA Ala-1 -D-Ala-AMPA des- 1-AMPA des-1,2-AMPA des-1,6-AMPA des-l&A Trp-3-Phe-AMPA Gln-4-Nle- AMPA Arg-6-Glu,Gly-7-Ala-AMPA Gly-7-Ala- AMPA Tyr-52-Phe-PLA2 Tyr-73-Phe-PLA2 ~~~~
RQY
00
(s-') PLAZ
270 0.8 270 230 270 120 9 25 17 200 180 230 250 290 290
1 2.13 1.04 1.86 1.88 1.84 2.64
