Hydrolysis of esters by carboxypeptidase A requires a penta-coordinate metal ion

Communication

THEJOURNAL OF BIOLOGICAL CHEMISTRY Vol. 257, No. 1, Issue of January 10, pp. 24-27, 1982 Printed in L! S.A .

Hvdrolvsis of Esters bv C&bo&peptidase A Ruequires a Penta-Coordinate Metal Ion*

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enzyme is displaced by the carbonyl oxygen of the inhibitor (1). On this basis, the Zn2+ion is generally thought to actonly as a Lewis acid in activating the carbonyl group of the substrate for nucleophilic attack. In contrast, we have demonstrated that ionization of a metal-bound water molecule is (Received for publication, September 10, 1981,and in revised form, required for breakdown of the mixed anhydride, acyl-enzyme October 19, 1981) intermediate formed in ester hydrolysis (2). These results Lawrence C. Kuo$ and Marvin W. Makinen8 have prompted us to conclude that the metal-bound water From the Department of Biophysics a n d Theoretical molecule is not displaced upon the binding of true substrates. Biology, Cummings Life Science Center, The University It is therefore necessary to show that the coordination enviof Chicago, Chicago, Illinois 60637 ronment of the metal ion in catalytically competent reaction intermediates differs fromthat in enzyme-inhibitorcomplexes. The catalytic role of the metalion in bovine carbox- For this purpose, we have carried out electron paramagnetic ypeptidase A (peptidyl-L-aminoacidhydrolase; EC resonance studies of the Co2+-reconstitutedenzyme to inves3.4.12.2) is investigated by application of cryoenzymo- tigate the local environment of the metal ion. Our results not logic andelectronparamagneticresonancemethods only confirmthat themetal-bound water molecule is retained with use of theCo2+-reconstitutedenzyme. Incorpora- in the acyl-enzyme intermediate but also demonstrate that tion of 1 7 0 into oxygen-donor ligands induces a substantial change in the spin-lattice relaxation probabil- the carbonyl oxygen of the substrate is coordinated to the metal ion. This observation requires that the metal ion is ity of the paramagnetic ion. While a change in spinaltered from a tetra-coordinate species in the free enzyme to lattice relaxation is observed forthe free Co2+-enzyme a penta-coordinate species in the acylenzyme reaction interin "0-enriched water, no change is observed for the enzyme complexed to glycyl-L-tyrosine. These results mediate. are consistent with x-raycrystallographic studies EXPERIMENTALPROCEDURES showing that the metal-bound water molecule in the The crystalline a-isozyme of bovine pancreatic ZnCPA' was used active site is displaced upon binding of the peptide as previously described (2,3) and converted to theCo"-reconstituted inhibitor.A change inspin-latticerelaxation of the Co2+ form according to the procedure of Latt and Vallee (4). Cryosolvent ion in the mixed anhydride, acyl-enzyme intermediate mixtures (5) were employed as previously described (2). formedwiththespecific ester substrate 0-(trans-pThe synthesis of O-(trans-p-chlorocinnamoyl-l-'70)-L-~-phenyllactate was achieved by preparation of 170-enrichedp-chlorocinnamic chlorocinnamoy1)-L-/l-phenyllactateis observed when acid. "0-enriched water (0.3 ml) (52.79% "0, 41.79% I8O; Monsanto 1 7 0 is enriched either into water or into the carbonyl Research Corporation, Miamisburg, OH) was added to the acid chlooxygen position of the scissile bond of the substrate. Since the proteinsupplies three amino acidside chains ride of p-chlorocinnamic acid (6) (0.4 g, 1.99 mmol) suspended in 20 ml of dry benzene and stirred for 4 days at room temperature in a as ligands to the metal ion,these results indicate that closed vessel. Upon evaporation of the solvent and drying over Pz05, the metalion is altered from a tetra-coordinate species white crystals (0.35g, 1.92 mmol, 96% yield) were recovered and then in the free enzyme to a penta-coordinate species in the converted to the acid chloride (0.18g, 0.90 mmol, 46% yield). The acyl-enzyme reaction intermediate. In addition, the re- acid chloride was condensed (6) with L-P-phenyllacticacid previously sults provide structural support for our assignment of dried over P205, and the ester product was recrystallized from benionization of a metal-bound water molecule in rate- zene-hexane (0.089 g, 0.27 mmol, 27% yield). Mass spectral analysis limiting deacylation (Makinen, M. W., Kuo, L. C., Dy- showed 14.2 g atom 76 enrichment of the carbonyl oxygen position mowski, J. J., and Jaffer, S. (1979) J. Biol. Chem 254, with I7O (theoretical limit, 21.5 g atom 76). Isotopic enrichment of Co(CH3C00)dmZand of the isomorphous 356-366) and affirm that the metal-hydroxide species is complex (7) with "0 was carried out by preparing "0-enriched the nucleophile responsible for the breakdown of the Zn2+ acetic acid. Acetic anhydride was dried over Pz05, refluxed, and mixed anhydride reaction intermediate of carboxypep- multiply distilled. The purified acetic anhydride (0.75 ml, 8 mmol) tidase A. was immediately reacted with I70-enriched water (0.3 m l ) in the presence of10p1of 6 N HCl (Baker Ultrex) at 40 "C for 2 days followed by refluxing for 6 h. Mass spectral analysis showed enrichment of CH,COOH with "0 to 27 g atom %. A slight excess of cobalt metal powder was suspended in 0.3 ml of I70-enriched acetic acid, refluxed for several hours, and then heated at -45 "C for several days to yield a red solution of Co(CH3COO)z. The supernatant was removed with a pipette with the reaction vessel placed over a small laboratory magnet to attract unreacted metal particles. The concentration ofCo" was determined by atomic absorption analysis. A similar procedure was used to prepare the "0-enriched Zn2' complex using 0.70 ml of the "0-enriched acetic acid. The unreacted zinc metal was removed by filtration, and Zn(CH3COOk was crystallized from ethanol that hadbeen dried and distilled over CaO just prior to use. These products were then employed to prepare (7) polycrystalline

In the carboxypeptidase A- (peptidyl-L-aminoacid hydrolase; EC 3.4.12.2) catalyzed hydrolysis of peptides and esters, the metal ion of the enzyme is assumed to remain tetrahedrally coordinated throughout the reaction. Difference Fourier syntheses of complexes of the enzyme with peptide inhibitors show that the metal-bound water molecule in the free * This work was supported by Public Health Service Grant GM 21900 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Predoctoral Student supported by Public Health Service Training Grant GM 07183 of the National Institutes of Health. 8 To whom to address correspondence; Established Investigator of the American Heart Association for part of the tenure of this investigation.

I The abbreviations used are: ZnCPA, Zn2+-containingor native carboxypeptidase A; ClCPL, 0-(trans-p-chlorocinnamoy1)-L-P-phenyllactate; CPA, carboxypeptidase A; CoCPA, Co2+-substituted carboxypeptidase A; c.w., continuous wave; Im, imidazole.

24

Carboxypeptidase Environment in IonMetal

A

25

Zn(CH3C00)21mz intowhich the corresponding Co2+ complex was incorporated to -0.2 mol fraction %. Glycyl-L-tyrosinewas obtained fromSigma.Imidazole was purchased from Aldrich and was three times recrystallized from water and dried beforeuse. EPR spectra were recorded at 100 kHz modulation and 9.4 GHz microwave irradiation witha Bruker ER200D spectrometer operated in the TElmmode and equipped with an Oxford Instruments ESRlO liquid helium cryostat (8). Progressive saturation curves of the resoa C.W. nanceabsorption of the Co2+-enzymeweredeterminedby saturation technique described fromthis laboratory (9). RESULTS AND DISCUSSION

+

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The first derivative EPR absorption spectrum of CoCPA is 1 I 1 I I I 1 1000 2000 3000 4000 illustrated in Fig. 1. The spectrum is that of a high spin Co2+ Magnet~cF d d (GI ion in a rhombically distorted environment with approximate FIG. 1. First derivative EPR absorption spectum of CoCPA. g values of5.94, 3.01, and 2.05. Thelineshaperemains M in a 0.25 of The enzyme was dissolved to a concentration of 3.7 X constant in the 5-12 K temperature range under conditions M sodium chloride solution buffered to pH 7.5 at 0 "C with 0.02 M nonsaturating microwave power. Also, noincreaseinline sodium cacodylate. The spectrum was recorded with 2 milliwatts of width is observed with saturatingmicrowave fields. Although incident microwave powerat 25 G modulation amplitude.No change we have carefully compared the spectrum of the Co2+-enzyme in line shape is observed with lower modulationamplitude. The (first derivative) resonance absorption signal amplitude in several regions in solutions of natural abundance water to the spectrum of of the spectrum was monitored as a function of incident microwave the enzyme in "0-enriched water, we have been unable to detect broadening due to the ligand hyperfine interactions of power. The saturation curve for the prominent peak at 1140 G at the lowest temperature attainable yields an estimate for PI/*greater than metal-bound H2170.The broad banded resonance absorption 30 milliwatts. Since the accuracy of determining PI/>is constrained features of the Co2+ion in theenzyme prevent a more precise by the asymptotic limit of the slope at high values of P, we have characterization of ligand hyperfine broadening. On the otherchosen the 1320-1740 G peak-to-peak amplitude centered at 1530 G hand, we have been able toconfirm2 the broadening influence to characterize 170-induced differential saturation effects for CoCPA. The differential saturation behavior of this region parallels that of of H2170 coordinated to the axial sixth position of the high spin Fe3+ ion in (aquo)metmyoglobin, as reported by others the peak at 1140 G inall other respects but allowsmoreprecise comparison of Pli2 values overthe entire 5-12 K range because lower (10, 11). power levelsare required forsaturation. On this basis, the normalized For the Co2+-substituted enzyme, as illustratedin the inset signal amplitude centered at 1530 G with increasing microwave power of Fig. 1,progressive C.W. saturation curveswere evaluated by (in milliwatts) is compared in the inset for CoCPA at 8 K in solvents according to containing (a)natural abundance H20 (0)and ( b ) 26 g atom % I7Oa method thatyields a graphical estimateof PlI2 defined by others (12, enriched H20 (A). The highest possible extent of isotopic enrichment the relation S / f i = (1 P/P1/2)-b/2 13). The parameterP112is themicrowave power a t which the was used. The intersection of the asymptotic limits of the slopes thesevaluesare 12.6and 2.5 (defined in Ref. 14) is defines(10, 11) the value of P1/2; saturationfactor (1 + y2H~2TlT2)" milliwatts fora and b, respectively. A t 10 K, there is no change in line equal to0.5 and servesas an index of the differential saturation shape and the estimated values of PI/>are correspondingly 18.0 and behavior of the paramagnetic species. With accurate control 3.0 milliwatts (data not shown). of the sample temperature as described (9), the graphically estimated values of log PlI2are reproducible to within +lo% broadening of the resonance absorption due to the ligand for independently prepared sets of samples in natural abundance and 170-enriched water. While a decrease in log by hyperfine interactions of the ( I = 5/2) 170nucleus. Unresolved a factor of -2.7 is observed in the presenceof H2I7O, there is hyperfine broadening is also observed for the high field resois increased by -15 no change in the value of b as indicated by the slope of the nance absorption. The peak-to-peak width ligand hyperfine coupplot a t high values of P. Also, the value of b remains invariant G, suggesting an approximate (average) ling equivalent to 9.3 G/"O hyperfine line. This is consistent with temperature throughout the5-12 K range. complexes With no changein either the parameterb or theline width, with the rangeof values observed inother metal ion (16, 17). the shift in Pll2 means that only the spin-lattice relaxation In Fig. 2C, a comparison is shown of the progressive C.W. probability of the Co2+ ion has been altered. There are two possible sets of conditions that may underlie these observa- saturation curves of the natural abundance and 170-enriched tions. If 1/Tz has not been alteredby introduction of an 170- complexes of Co2+ based on the change in the peak-to-peak P1/2cc l/y2TlT2 = constant/T1. signal amplitude of the low field resonance absorption with ligand, it is apparent that Alternatively, Hyde (15) has pointed out that thesignal am- increasing microwave power. There is a decrease in log PIr2 by a factor of 1.5 upon incorporation of 27 g atom % I7O into plitude is proportionalto only l/Tl for inhomogeneously broadened resonance absorptionif T1> T2>> l/yHl. In either the acetateligand of the Coz+complex. The differential satucase, the value of PI12 remains proportional toonly the spin- ration behavior remained parallel to thatin Fig. 2C throughout the 5-10 K range with no changein the parameter b and lattice relaxation probability designatedas l/Tl. no changein the peak-to-peak line width for either the natural In similar studies, we havecomparedthe influence of 17 0 on the progressive C.W. saturation properties of abundance or 170-enriched complexes. Underthese condiCo(CH&00)21m2. Thefirst derivative EPR absorption spec- tions, the shift in P 1 / 2 again means that only the spin-lattice trum of C O ( C H & O O ) ~incorporated I~~ into the host matrix relaxation probability of the Co2+ion has been altered. The spectrumof the inhibitorcomplex formed with glycylof the Zn2+ complex shown in Fig. 2A is identical with that L-tyrosine is shown in Fig. 3. In contrast to the freeenzyme, reported by Horrocks et al. (7). InFig. 2B, the low field region there is no change in P1/2 for the Co2+ ion in the enzymeof the spectrum of Co(CH3C00)21m2 enriched to27 g atom inhibitor complexformedin the presence of H2I70. These % with I7O is compared to that in Fig. 2A to illustrate the results are consistent with the results of x-ray crystallographic studies. Difference Fourier studies have demonstrated that M. W. Makinen, M. B. Yim, and L. C. Kuo, unpublished observations. the metal-bound water molecule in CPA is displaced upon

IonMetal

26

A

Carboxypeptidase Environment in

the presence of the '70-enriched substrate results in an increase inlogby a factor of -1.7. The opposite change in P1pwhen "0 is incorporated into the substrate indicates that the alteration cannot be ascribed to hydrolysis of the substrate with subsequent exchange of I7O into bulk water. In parallel studies in this laboratory, we have observed that the saturation behavior of the high spin metalcationsin (aquo)metmyoglobin, (aquo)methemoglobin, and Co'+-reconstituted horse liver alcohol dehydrogenase is also influenced by the presence of H2170 and that thepresence of H,170 can lead either to a decrease or an increase in P 1 / 2 depending upon the complex prepared.' For each of these metalloproteins, a

1

2000 Mognetlc Field

1000

4000

3000

(GI

I

I

I

1300

1600

Magnetic Field (G)

1900

-4

1

log P

FIG. 2. First derivativeEPR absorption spectrumand power

I

I

I

1000

2000

I

I

I

3000

Magnellc Fleld IG)

FIG. 3. Firstderivative

EPR absorptionspectrum of the complex of CoCPA with glycyl-L-tyrosine. The enzyme-inhibitor

saturation behavior of Co(CH3COO)zImZ incorporated into the complex wasprepared with a 4-fold molar excess of the inhibitor. The host matrix of the isomorphous polycrystalline Zn2+complex. spectrum was recorded with 1milliwatt of microwave powerincident In A is shown the spectrum of the natural complex while in B the on the cavity. The inset compares the C.W. microwave power saturaspectrum of the "0-enriched complex (broken line) in the low field tion behavior of the resonance absorption at lo00 G for the Co" ion

-

4.49 is compared to that of the natural region centered at g abundance complex (solid line). The acetate was enriched to 27 g atom % with I7O for both the Co2+complex and for the host matrix of the Zn2+ complex and identical amounts (0.120g) of natural and isotopically enriched materials were employed for collecting saturation data. In B, the peak-to-peak amplitude has been made approximately equal by adjustment of the gain. The modulation amplitude was 8 G. In C is compared the microwave power saturation behavior of the "0-enriched complex (A) to the natural abundance (8)sample. Although crystallization of the natural and I70-enriched complexes was carried out under similar conditions, the actual content of Co2' was determined by atomic absorption methods to be 0.19 molfraction % for the natural abundance complex and 0.24 mol fraction 76 for the '70-enriched complex. Nonetheless, despite the slightly higher number of paramagnetic sites, the influence of I7O is to decrease PI,Zwith no change in the asymptotic limit of the slope at high values of P. At 5 K, the values of P,/2are 44.7 and 12.6 milliwatts for the natural abundance and I70-enrichedcomplexes whileat 9 K (data not shown), the corresponding values of P,/2 are 81.3 and 47.0 milliwatts.

in the enzyme-inhibitor complex prepared in the presence of natural abundance water (0)and 26 g atom % "0-enriched water (0).The graphically estimated value of P1,2 is 1.6 milliwatts at 6 K and 3.2 milliwatts at 8 K. The weak signals at 1540 and 3250 G are due to paramagnetic inpurities in the quartz sample tube. AU other conditions as in Fig. 1.

1

n

log P

1000 2000 3000 4000 binding of the inhibitor glycyl-L-tyrosine (1).Since not all of Mognetu F d d (GI the water molecules in the active site of CPA are displaced by FIG. 4. First derivative EPR absorption spectrum of the the binding of inhibitors (l),the results in Fig. 3 also suggest that long range interactions between the Co" ion and distant acyl-enzyme intermediate of CoCPA formed with ClCPL. The solvent molecules cannot account for the change in the satu- preparation of the intermediate hasbeen described previously (2) and was carried out directly in the EPR quartz sample tube at -70 "C. ration behavior of the Co2+ion in CoCPA. The resultant cryosolvent mixture corresponds to anethylene glycol/ The EPR spectrum of the acyl-enzyme intermediate of methanol/water (402040, v/v) mixture containing 3 X M CoCPA CoCPA formed with ClCPL and stabilized (2) under subzero and 1 X M substrate in a 0.25 M sodium chloride solution buffered temperature conditions is illustrated in Fig. 4. The spectrum to pH 7.5 with 0.02 M sodium cacodylate at -60 "C. The inset is distinct from that of free CoCPA. In the inset of Fig. 4, we compares the C.W. power saturation behavior of the resonance abhave compared the differential saturation behavior of the sorption at 1100 G for the reaction intermediate at 7 K in the presence (a)natural abundance water and substrate (0);( b ) 26 g atom % acyl-enzyme intermediate under conditions of selective incor- of I70-enriched water (0); and (c) 14.2 g atom % "0-enriched substrate poration of I7O either into the solvent or into the carbonyl (A). The values of PI/*estimated at 7 and 9 K are, respectively, 0.08 oxygen of the substrate. In the presence of H2170, a decrease and 0.44 milliwatts for a;0.04 and 0.27 milliwatts for b; and 0.56 and 1.98 milliwatts for c. Other conditions are as in Fig. 1. in log by a factor of -1.3 is observed. On the other hand,

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I

Carboxypeptidase Environment in IonMetal

, / K . 1 - , . ;HC)"COi.

Glu-270 -C-O-C-R

FIG. 5. Schematic drawing of the structure of the metal ion environment in the acyl-enzyme intermediate of CPA formed with ClCPL.

27

studies in which only a tetra-coordinate Zn2+ ionis invoked. For small molecule metal ion complexes that catalyze the hydrolysis of esters, anhydrides, and peptides, metal-hydroxide nucleophilic attack is the kinetically preferred pathway in those complexes where both a water molecule or hydroxide anion are coordinated to the metal ion together with the carbonyl oxygenof the scissilebond (26). On this basis, detection of a metal-coordinated water molecule inthe pentacoordinate reaction intermediate of CPA affirms that deacylation of the mixed anhydride intermediate proceeds via attack on the carbonyl carbon of the substratedirectly by the potent metal-hydroxide nucleophile. Acknowledgments-We thank T. R. Koch and Dr. M. B. Yim for assistance in the synthesis of '70-enriched ClCPL and in the use of the liquid helium cryostat. REFERENCES 1. Hartsuck, J. A., andLipscomb, W. N. (1971) in The Enzymes (Boyer, P. D.,ed) Vol. 111, pp. 1-56, Academic Press, New York 2. Makinen, M. W., Kuo, L. C., Dymowski, J. J., and Jaffer, S. (1979) J. Bwl. Chem. 254,356-366 3. Makinen, M. W., Yamamura, K., and Kaiser, E. T. (1976) Proc. Natl. Acad. Sci. U. S. A . 73, 3882-3886 4. Latt, S. A,, and Vallee, B. L. (1979) Biochemistry 10, 4263-4270 5. Douzou, P., Hui Bon Hoa, G., Maurel, P., and Travers, F. (1976) in Handbook of Biochemistry and Molecular Biology (Fasman, G. D., ed) 3rd ed, pp.520-540,ChemicalRubber Publishing Co., Cleveland, OH 6. Suh, J., and Kaiser, E. T. (1976) J. Am. Chem. SOC.98,1940-1947 7. Horrocks, W.Dew., Jr., Ishley, J. N., Holmquist, B., and Thompson, J. S. (1980) J. Inorg. Biochem. 12, 131-141 8. Campbell, S. J., Herbert, I. R., Warnick, C. B., and Woodgate, J. M. (1979) Reu. Sci. Instrum. 47,11761177 9. Yim, M. B., Kuo, L. C., and Makinen, M. W. (1982) J. Magn. Resonance 46, in press 10. Vuk-Pavlovii.,S., and Siderer, Y. (1977) Biochem. Biophys. Res. Commun. 79,885-889 11.Gupta, R. K., Mildvan, A. S., and Schonbaum, G. R. (1979) Biochem. Biophys. Res. Commun. 89, 1334-1340 12. Beinert, H., and Orme-Johnson, W. H. (1967) in Magnetic Resonance in Biological Systems (Ehrenberg, A,, Malmstrom, B. G., and Vanngird, T., eds) pp. 221-247, Pergamon Press, Oxford 13.Rupp, H., Rao, K. K., Hall, D. O., andCammack, R. (1978) Biochim. Biophys. Acta 537,255-269 14. Poole, C. P., Jr. (1967) Electron Spin Resonance. A Comprehensive Treatise on Experimental Techniques,pp. 695-733,Wiley, New York 15. Hyde, J. S. (1960) Phys. Reu. 119, 1492-1494 16. Getz, D., and Silver, B. L. (1974) J. Chem. Phys. 61, 630-637 17. Melamud, E., and Silver, B. L. (1973) J. Phys. Chem. 77, 18961900 18.Abragam, A., and Bleaney, B. (1970) Electron Paramagnetic Resonance of Transition Zons, p. 560, Pergamon Press, Oxford 19. Baker, J. M., and Ford, N. C., Jr. (1964) Phys. Reu. 136A, 16921701 20. Larson, G. H., and Jeffries, C. D. (1966) Phys. Rev. 145, 311-324 21. Denisenko, G. A., and Khaimovich, E. P. (1971) Sou. Physics Solid State 13, 1539-1540 22. Argas, P., Garavito, R. M., Eventoff, W., Rossmann, M. G., and Brinden, C. I. (1978) J. Mol. Biol. 126, 141-158 23. Kaiser, E. T., and Kaiser, B. L. (1972) Accts. Chem. Res. 5, 219224 24. Breslow, R., andWernick, D. L. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 1303-1307 25. Lipscomb, W. N. (1974) Tetrahedron 30, 1725-1732 26. Buckingham, D. (1977) in Biological Aspects ofInorganicChemistry (Dolphin, D., ed) pp. 141-196, Wiley, New York

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metal-bound water molecule has been identified by x-ray crystallographic studies. The introduction of a metal coordinated H2l7Omolecule influences only the electronic spin-lattice relaxation probability in these metalloproteins since no alteration is seen in the parameter b. In contrast to the influence of '70-enriched water, no change in P I / Zis observed in the presence of H2180.The lack of an effect with Hz"0 and the variety of paramagnetic metal ion complexesin which the effect is observedindicates that the perturbation is magnetic in origin and requires coordination of the metal ion by an I 7 O enriched oxygen-donor ligand. For paramagnetic materials in which the nuclear neighbors belong to more than one isotopic species, the precessing magnetization of the sample is given by a weighted sum of contributions due to each type of electron-nuclear pair. Thus, the direction and extent of the observed shift in of an "0-enriched sample will depend upon both the factor by which the intrinsic spin-lattice relaxation probability of the metal ion-"O pairs is greater or smaller than thatof the metalion-I60pairs and the proportion of metal ion-I70pairs. At present, there is no explicit theory to account quantitatively for the influence of an 1 7 0 ligand on the electronic spinlattice relaxation of a paramagnetic metal ion. The influence of isotropic nuclear hyperfine magnetic fields of paramagnetic ions on electronic spin-lattice relaxation rates has been described (18-21), but the theory has not been extended to evaluate the possible effects of anisotropic ligand hyperfine interactions that are dependent upon coordination geometry. For this reason, we employ the shift in P 1 / 2 only as an empirical spectroscopic probe to identify the chemical origins of oxygen-donor ligands to the active site metal ion in the acyl-enzyme reaction intermediate of CPA. We have previously assigned the ionization required for rate-limiting breakdown of the acyl-enzyme intermediate of CPA to a metal-bound water molecule in both the Zn2+and Co2+-enzymes(2). The data in Fig. 4 demonstrate that both a solvent molecule and the carbonyl oxygen atom of the substrate arecoordinated to themetal ion in the mixed anhydride intermediate and thereby provide direct structural support for this assignment. Since the protein donates three amino acid side chains to the metal ion (l),these results indicate that the active site metal ion with two oxygen-donor ligands must be penta-coordinate in the mixed anhydride reaction intermediate. Indeed, a penta-coordinate Zn2+ion with a metal-bound water molecule is compatible with the steric constraints of the active site of CPA (22). The coordination environment of themetal ionin the reaction intermediate based on the results of our EPR studies is schematically illustrated in Fig. 5. It differs markedly from that assumed for interpretations of the mechanism of CPA derived through chemical (23,24) and model building (1,25)

A