Ionization States of the Complex Formed Between 2-benzyl-3-phosphonopropionic Acid and Carboxypeptidase A

Overview

Journal Biochem J

Specialty Biochemistry

Date 1988 Sep 15

PMID 3196297

Citations 1

Authors

U B Goli

D Grobelny

R E Galardy

Affiliations

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Abstract

The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.

Citing Articles

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References

Christianson D, Lipscomb W . Binding of a possible transition state analogue to the active site of carboxypeptidase A. Proc Natl Acad Sci U S A. 1985; 82(20):6840-4. PMC: 390783. DOI: 10.1073/pnas.82.20.6840. View

Gardell S, Craik C, Hilvert D, Urdea M, Rutter W . Site-directed mutagenesis shows that tyrosine 248 of carboxypeptidase A does not play a crucial role in catalysis. Nature. 1985; 317(6037):551-5. DOI: 10.1038/317551a0. View

Mock W, Tsay J . A probe of the active site acidity of carboxypeptidase A. Biochemistry. 1986; 25(10):2920-7. DOI: 10.1021/bi00358a028. View

Gettins P . On the coordination of inhibitors to the metal ion of carboxypeptidase A. A 113Cd and 31P NMR study. J Biol Chem. 1986; 261(33):15513-8. View

Hirose J, Ando S, KIDANI Y . Excess zinc ions are a competitive inhibitor for carboxypeptidase A. Biochemistry. 1987; 26(20):6561-5. DOI: 10.1021/bi00394a041. View

Simpson R, Riordan J, Vallee B . FUNCTIONAL TYROSYL RESIDUES IN THE ACTIVE CENTER OF BOVINE PANCREATIC CARBOXYPEPTIDASE A. Biochemistry. 1963; 2:616-22. DOI: 10.1021/bi00903a039. View

Navon G, Shulman R, WYLUDA B, Yamane T . Nuclear magnetic resonance studies of the active site of carboxypeptidase A. Proc Natl Acad Sci U S A. 1968; 60(1):86-91. PMC: 539134. DOI: 10.1073/pnas.60.1.86. View

Schechter I, Berger A . On the active site of proteases. 3. Mapping the active site of papain; specific peptide inhibitors of papain. Biochem Biophys Res Commun. 1968; 32(5):898-902. DOI: 10.1016/0006-291x(68)90326-4. View

Lipscomb W, Hartsuck J, Reeke Jr G, Quiocho F, Bethge P, Ludwig M . The structure of carboxypeptidase A. VII. The 2.0-angstrom resolution studies of the enzyme and of its complex with glycyltyrosine, and mechanistic deductions. Brookhaven Symp Biol. 1968; 21(1):24-90. View

10.

Navon G, Shulman R, WYLUDA B, Yamane T . Nuclear magnetic resonance study of the binding of fluoride ions to carboxypeptidase A. J Mol Biol. 1970; 51(1):15-30. DOI: 10.1016/0022-2836(70)90266-4. View

11.

Byers L, Wolfenden R . Binding of the by-product analog benzylsuccinic acid by carboxypeptidase A. Biochemistry. 1973; 12(11):2070-8. DOI: 10.1021/bi00735a008. View

12.

Cushman D, Cheung H, SABO E, ONDETTI M . Design of potent competitive inhibitors of angiotensin-converting enzyme. Carboxyalkanoyl and mercaptoalkanoyl amino acids. Biochemistry. 1977; 16(25):5484-91. DOI: 10.1021/bi00644a014. View

13.

Biryukov A, Ishmuratov B, Khomutov R . Transition-state analogues of aminoacyl adenylates. FEBS Lett. 1978; 91(2):249-52. DOI: 10.1016/0014-5793(78)81184-3. View

14.

Makinen M, Kuo L, Dymowski J, Jaffer S . Catalytic role of the metal ion of carboxypeptidase A in ester hydrolysis. J Biol Chem. 1979; 254(2):356-66. View

15.

ONDETTI M, CONDON M, Reid J, SABO E, Cheung H, Cushman D . Design of potent and specific inhibitors of carboxypeptidases A and B. Biochemistry. 1979; 18(8):1427-30. DOI: 10.1021/bi00575a006. View

16.

Tipton K, Dixon H . Effects of pH on enzymes. Methods Enzymol. 1979; 63:183-234. DOI: 10.1016/0076-6879(79)63011-2. View

17.

Rees D, Lipscomb W . Binding of ligands to the active site of carboxypeptidase A. Proc Natl Acad Sci U S A. 1981; 78(9):5455-9. PMC: 348764. DOI: 10.1073/pnas.78.9.5455. View

18.

Palmer A, Ellis P, Wolfenden R . Extreme state of ionization of benzylsuccinate bound by carboxypeptidase A. Biochemistry. 1982; 21(20):5056-9. DOI: 10.1021/bi00263a032. View

19.

Vallee B, Galdes A . The metallobiochemistry of zinc enzymes. Adv Enzymol Relat Areas Mol Biol. 1984; 56:283-430. DOI: 10.1002/9780470123027.ch5. View

20.

Grobelny D, Goli U, Galardy R . 3-Phosphonopropionic acids inhibit carboxypeptidase A as multisubstrate analogues or transition-state analogues. Biochem J. 1985; 232(1):15-9. PMC: 1152831. DOI: 10.1042/bj2320015. View