Rate-determining Step in Phospholipase A2 Mechanism "0 ISOTOPE EXCHANGE DETERMINED BY I3C NMR*

Rate-determining step in phospholipase A2 mechanism. 18O isotope exchange determined by 13C NMR. Hi80 isotope exchange into specifically 13C-labeled substrate was used to obtain information on the rate- limiting step in the action of the phospholipase A2 from the venom of the Indian cobra (Nuju nuju nuju). Incor- poration of "0 was detected by the effect of "0 on 13C chemical shifts in 13C NMR. The enzymatic hydrolysis of a micellar phosphatidylcholine analogue of platelet- activating factor 1-alkyl-2-[ l-'3C]lauroyl-sn-glycero-3-phosphorylcholine proceeds by an 0-acyl cleavage of the sn-2 ester bond. The reaction was examined for simultaneous "0 incorporation into the substrate. No exchange was found, suggesting that the hydrolytic step is not followed by a higher energy transition state and that it or a step before it appears to be rate- limiting. Previous experiments on phosphatidylethanolamine activation indicate that kcat is altered but that the k, remains the same upon activation, suggesting that the binding steps occurring before the hydrolytic step are not affected. This strongly suggests that the hydrolytic step is in fact the rate-limiting step under these conditions. The 13C,'80 NMR technique should be generally applicable to mechanistic questions of this type.

H i 8 0 isotope exchange into specifically 13C-labeled substrate was used to obtain information on the ratelimiting step in the action of the phospholipase A2 from the venom of the Indian cobra (Nuju nuju nuju). Incorporation of "0 was detected by the effect of "0 on 13C chemical shifts in 13C NMR. The enzymatic hydrolysis of a micellar phosphatidylcholine analogue of plateletactivating factor 1-alkyl-2-[ l-'3C]lauroyl-sn-glycero-3-phosphorylcholine proceeds by an 0-acyl cleavage of the sn-2 ester bond. The reaction was examined for simultaneous "0 incorporation into the substrate. No exchange was found, suggesting that the hydrolytic step is not followed by a higher energy transition state and that it or a step before it appears to be ratelimiting. Previous experiments on phosphatidylethanolamine activation indicate that kcat is altered but that the k, remains the same upon activation, suggesting that the binding steps occurring before the hydrolytic step are not affected. This strongly suggests that the hydrolytic step is in fact the rate-limiting step under these conditions. The 13C,'80 NMR technique should be generally applicable to mechanistic questions of this type.
It is now well established that pancreatic and venom phospholipase A2 display a marked preference for micellar substrates and act poorly on monomeric substrate (for review, see Ref. 1). Over the years, at least four explanations have been advanced for the requirement of phospholipase A2 for aggregated substrate which can be summarized as follows: (i) the physical state of the substrate phospholipid is altered in going from monomer to micelle (substrate effect); (ii) the enzyme undergoes a conformational change after it binds micellar phospholipid (enzyme effect); (iii) the higher local concentration of substrate in the micelle brings the enzyme closer to saturation (reaction effect); and (iv) product removal is the rate-limiting step and is facilitated by the micelle (product effect). A detailed consideration of these possibilities can be found elsewhere (1). Our previous studies have focused on the first three possibilities. We have described changes in substrate conformation in going from monomer to micelle (2) and conformational changes in the enzyme upon binding certain phospholipids leading to enzyme aggregation (3-7). We have also shown that substrate saturation on the enzyme * This work was supported by National Science Foundation Grant DMB 85-18684 and National Institutes of Health Grant GM 20501. Work performed at the Southern California Regional NMR Facility at the California Institute of Technology was supported by National Science Foundation Grant CHE 84-40137. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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is not increased in the presence of certain activators (8-10).
We now wish to report initial studies eventually aimed at examining the merit of the proposal that the product release step is slow in the reaction of phospholipase A*, but is accelerated when the enzyme shows higher specific activities. In this paper, we have determined the rate-limiting step for the micellar system (e.g. in the presence of certain activator phospholipids and aggregated phospholipids). For this purpose, 13C NMR was employed to quantitate the degree of Hi80 exchange into a suitably 13C-labeled substrate and product during reaction with phospholipase A2. Advantage was taken of the "0 isotope effect on the I3C chemical shifts established by Risley and Van Etten (11,12). Exchange reactions have been studied using this technique with 13C NMR (13, 14) and 31P NMR (15,16). In this initial study, we used a phosphatidylcholine (PC') analogue which is a good substrate for the enzyme. It was labeled with in the sn-2 ester position and contains an alkyl ether instead of an ester in the sn-1 position so as to eliminate all ambiguities in the assignment of resonances, especially the possibility of acyl migration (17) which could have complicated the NMR results.
Tentative 'H NMR assignments for lauroyl-PAF in CDC13 were made using one-and two-dimensional NMR. Spectra were recorded at 360.7 MHz on a homebuilt spectrometer employing a Nicolet 1280 computer. J-Correlated spectra (20) were obtained using the following conditions. Spectral width was +lo00 Hz; 1 K memory was used to accumulate 512 spectra (incremented evolution period). The assignment is as follows: 0.90 ppm of w-CH3 (acyl + alkyl chain), 1.28 ppm of (CHS)" (acyl + alkyl chain), 1. X-100. The activity of the cobra venom phospholipase A2 toward lauroyl-PAF was about the same as that toward dipalmitoyl-PC (18). The concentration of phospholipase A2 was adjusted to obtain the desired amount of hydrolysis during the incubation time (generally 12-20 h). The isotope effect on the I3C chemical shift is quite small and requires that extremely narrow line widths be quantitated. Although it would be desirable to follow the enzymatic reaction directly in the NMR spectrometer while it is proceeding, the compounds of interest tend to form aggregates in aqueous solution as well as in some organic solvents (resulting in broad lines) and must be extracted first so as to be examined in CHClJCH,OH. Thus, at the end of the incubation, the reaction was quenched by the addition of EDTA (50 mM final concentration), which, by chelating the Ca2+, both inhibits the enzyme and makes the lauric acid completely extractable by the Bligh and Dyer method (21). The residual substrate and fatty acid were extracted with CHC13/CH30H/acetic acid

RESULTS AND DISCUSSION
Assignment of 13C Spectra-The phospholipase Az hydrolyzes the fatty acid ester bond at the sn-2 position of phospholipids leading to free fatty acid (1). Thus, the 13C NMR chemical shift of [l-'3C]lauroyl-PAF was determined along with that of a free fatty acid used as a control ([l-'3C]palmitic acid), as shown in Fig. 1. The chemical shifts determined in CDC13:CH30H (5050, v/v) were 173.30 and 176.17 ppm, respectively, for the substrate and the product of the hydrolysis. These values agree with those reported in the literature (24). It should be noted that the chemical shift of the carboxylic acid carbon atom is very sensitive to many solution parameters, including its concentration (24).
Lauroyl-PAF was incubated with sufficient cobra venom phospholipase AP to obtain almost complete hydrolysis in 12 h. One incubation was carried out in the presence of Hi60 and a second one in the presence of Hi80. Both samples were examined after extraction; at 50 MHz, each gave only a single peak (line width about 3 Hz). When mixed together and examined at 126 MHz, two peaks separated by 0.028 ppm (about 3.5 Hz) were found (Fig. IC). The downfield peak (176.169 ppm) is assigned to the free fatty acid containing "0, whereas the upfield peak (176.141 ppm) is assigned to the free fatty acid in which an "0 is introduced (see below), consistent with the expected isotope effect (14). The magnitude of the upfield shift (0.028 ppm) is characteristic of a carboxylic acid (acetic acid) containing a single "0 label (13, 25).
Without sufficient EDTA, the Ca2+ salt of lauric acid can be extracted leading to a broad peak (Au, about 50 Hz) a t 182.22 ppm, consistent with the resonance of a carboxylic anion (24) and most probably due to the formation of reversed micelles in the apolar solvent. When properly treated with EDTA and extracted, the carboxylic acid peak always appears sharp with the expected chemical shift. In an experiment in which two peaks were observed for the lauric acid after incubation in H:'O/HisO mixtures, the sample was checked and the peaks were found to still be present after filtration over Chelex 100. Also some resonances coming from co- extracted Triton X-100 could be observed. Nevertheless assignments in Triton X-100 showed that no resonance appears downfield of 160 ppm (26); this demonstrates that the two resonance lines of [l-13C]lauric acid cannot be the result of the spectrum of the Triton X-100 present. All of these controls demonstrate that the two peaks for the lauric acid resonance after incubation in the presence of 180/1fi0 water are indeed the result of the isotope effect of "0 on the 13C nuclear shielding.
HiRO Exchange Studies with Phospholipid-If the enzymatic hydrolysis of diacyl-PC occurs by an 0-acyl cleavage, the reaction in the presence of 180-labeled water (Hz. , with indicating "0-label) should occur according to Equation 1. Thus, the production of '80-labeled lauric acid as examined by 13C NMR demonstrates that the hydrolysis of diacyl-PC by cobra venom phospholipase Az proceeds via the expected 0-acyl cleavage, as was found with the enzyme from Crotalus adamenteus venom by traditional methods employing mass spectroscopy (27). If exchange of Hi'0 occurs and if the two oxygens on the free fatty acid product are equivalent (28) (29), exchange would, therefore, only be expected if a transition state after the breakdown of the tetrahedral intermediate (such as a conformation change or product release) was dominant. Exchange also requires the equivalence of the two oxygens of the bound carboxylic acid product. Exchange could also occur by complete resynthesis from dissociated products, but resynthesis would not be expected to be observable because of the extremely exergonic external equilibrium constant. The hydrolysis of the lauroyl-PAF was carried out to about 50% completion in 50:50 H:'O/HiSO as solvent. The products of the hydrolysis were extracted, and the spectrum was recorded at 126 MHz. As shown in Fig. 2, the peaks obtained for the free fatty acid resonance at 176.515 ppm are resolved into two peaks separated by 0.024 ppm, whereas a sharp peak is obtained for the residual [l-'3C]lauroyl-PAF a t 173.73 ppm. Small differences in the chemical shifts from Fig. 1 are due to solvent, temperature, and concentration differences (24, 30).
If a transition state after the breakdown of a tetrahedral intermediate were dominant, the H4'0 would be incorporated into the substrate (RCOOR') by the reverse reaction, and after prolonged incubation, then an additional "0 label would also appear in the fatty acid product (RCOOH) due to the cleavage of substrate which was exchanged over time. A third peak in the fatty acid resonance region would then be expected because of the additivity of the nuclear shielding (14, 25, 28). For acetic acid at pH 8, the resonance peak for one "0 is at 0.027 ppm upfield from the l60 peak and the peak for two lRO labels incorporated appears 0.054 ppm upfield (12). For esters, "0 incorporation into the carbonyl gives a peak 0.038 ppm upfield (14). However, in our case, substrate labeling was not observed during the time of substrate hydrolysis nor was double labeling of the free fatty acid observed.
To eliminate nonenzymatic "0 exchange into the free fatty acid product as a cause of the observed spectra, the following control was carried out. [l-'3C]Palmitic acid (10 mM) was incubated for 24 h at 40 "C under the conditions described for lauroyl-PAF hydrolysis by phospholipase A, except that EDTA was added at the beginning of the incubation to ensure complete solubilization of palmitic acid. At the end of the incubation, [ l-'3C]palmitic acid was extracted and treated as described for the products of the enzymatic catalysis. When examined at 126 MHz, [1-':'C]palmitic acid gave one sharp peak, indicating that "0-labeled [l-13C]palmitic acid was not present and that no exchange occurs between the free carboxylic acid liberated by the enzyme and the Hi80 solvent under assay conditions. Implications for Rate-determining Step-These experiments show that there is no exchange of Hi80 back into the substrate ester. This argues against the presence of a transition state (such as a conformational change or product release) after the hydrolytic step of higher energy than that of the tetrahedral transition state for the substrate tested. We would not expect an enzyme that catalyzes a very exergonic reaction such as ester hydrolysis to bind the products so tightly that their release is the most energetically unfavorable process in the overall free energy profile (31); and indeed, such is ruled out by the present experiments.
Cobra venom phospholipase A, hydrolysis of poor substrates is activated by phosphorylcholine-containing phospholipids (3)(4)(5)10). Kinetic analysis of the long chain phospholipid mixed micelle system has shown that the activation is due to a change in kc,, whereas substrate binding (K,) is unchanged. Whereas these data had to be obtained with thiol analogues of phospholipids for reasons discussed elsewhere (8-lo), the analogues behave identically in every respect examined, and we believe that this result most likely holds for natural phospholipids as well. The results presented herein indicate that the step limiting kceat/km occurs at or before the hydrolytic step, and the kinetic results with activators showing no change in k , indicate that the binding steps occurring before the hydrolytic step are not affected. Therefore, the step limiting kcat (the rate determining step) is identical to the step limiting kCat/k, and thus also occurs a t or before hydrolysis. In conclusion, the hydrolytic step appears to be the ratedetermining step for cobra venom phospholipase Az hydrolysis of micellar phospholipids.
It should be pointed out that the studies reported herein were carried out on a good substrate that behaves similarly to PC. We must now carry out Hi80 exchange studies on monomeric substrates below the critical micellar concentration and phosphatidylethanolamine substrates in the absence of