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. 1996 Jul 1;317(Pt 1):35–40. doi: 10.1042/bj3170035

Determination of the ionization state of the active-site histidine in a subtilisin-(chloromethane inhibitor) derivative by 13C-NMR.

T P O'Connell 1, J P Malthouse 1
PMCID: PMC1217482  PMID: 8694783

Abstract

Subtilisin BPN' has been alkylated using benzyloxycarbonyl-glycylglycyl[1-13C]phenylalanylchloromethane+ ++. Using difference 13C-NMR spectroscopy a single signal due to the 13C-enriched alpha-methylene carbon of the subtilisin-(chloromethane inhibitor) derivative was detected. No evidence for the denaturation/ autolysis of this derivative was obtained from pH 3.5 to 11.5. However, incubating at pH 12.75 or heating in the presence of SDS at pH 6.9 did denature this derivative. The negative titration shift of the alpha-methylene carbon of the denatured derivatives confirmed that the inhibitor had alkylated N-3 of the imidazole ring of the active-site histidine. The positive titration shift of 3.96 p.p.m. and the pKa of 7.04 obtained from studying the native subtilisin-(chloromethane inhibitor) derivative are assigned to oxyanion formation. We conclude that the pKa of the alkylated histidine residue in the native subtilisin-(chloromethane inhibitor) derivative must be > 12 and that subtilisin preferentially stabilizes the zwitterionic tetrahedral adduct consisting of the oxyanion and the imidazolium ion of the active-site histidine residue. We show that even before the oxyanion is formed the pKa of the active-site histidine must be much greater than that of the oxyanion in the zwitterionic tetrahedral adduct. We discuss the significance of our results for the catalytic mechanism of the serine proteinases.

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Selected References

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  1. Bachovchin W. W. 15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. Biochemistry. 1986 Nov 18;25(23):7751–7759. doi: 10.1021/bi00371a070. [DOI] [PubMed] [Google Scholar]
  2. Bachovchin W. W. Confirmation of the assignment of the low-field proton resonance of serine proteases by using specifically nitrogen-15 labeled enzyme. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7948–7951. doi: 10.1073/pnas.82.23.7948. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bycroft M., Fersht A. R. Assignment of histidine resonances in the 1H NMR (500 MHz) spectrum of subtilisin BPN' using site-directed mutagenesis. Biochemistry. 1988 Sep 20;27(19):7390–7394. doi: 10.1021/bi00419a033. [DOI] [PubMed] [Google Scholar]
  4. Cleland W. W., Kreevoy M. M. Low-barrier hydrogen bonds and enzymic catalysis. Science. 1994 Jun 24;264(5167):1887–1890. doi: 10.1126/science.8009219. [DOI] [PubMed] [Google Scholar]
  5. Cleland W. W. Low-barrier hydrogen bonds and low fractionation factor bases in enzymatic reactions. Biochemistry. 1992 Jan 21;31(2):317–319. doi: 10.1021/bi00117a001. [DOI] [PubMed] [Google Scholar]
  6. Coggins J. R., Kray W., Shaw E. Affinity labelling of proteinases with tryptic specificity by peptides with C-terminal lysine chloromethyl ketone. Biochem J. 1974 Mar;137(3):579–585. doi: 10.1042/bj1370579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Finucane M. D., Hudson E. A., Malthouse J. P. A 13C-n.m.r. investigation of the ionizations within an inhibitor--alpha-chymotrypsin complex. Evidence that both alpha-chymotrypsin and trypsin stabilize a hemiketal oxyanion by similar mechanisms. Biochem J. 1989 Mar 15;258(3):853–859. doi: 10.1042/bj2580853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Finucane M. D., Malthouse J. P. A study of the stabilization of tetrahedral adducts by trypsin and delta-chymotrypsin. Biochem J. 1992 Sep 15;286(Pt 3):889–900. doi: 10.1042/bj2860889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frey P. A., Whitt S. A., Tobin J. B. A low-barrier hydrogen bond in the catalytic triad of serine proteases. Science. 1994 Jun 24;264(5167):1927–1930. doi: 10.1126/science.7661899. [DOI] [PubMed] [Google Scholar]
  10. Jordan F., Polgar L., Tous G. Proton magnetic resonance studies of the states of ionization of histidines in native and modified subtilisins. Biochemistry. 1985 Dec 17;24(26):7711–7717. doi: 10.1021/bi00347a031. [DOI] [PubMed] [Google Scholar]
  11. Malthouse J. P., Primrose W. U., Mackenzie N. E., Scott A. I. 13C NMR study of the ionizations within a trypsin-chloromethyl ketone inhibitor complex. Biochemistry. 1985 Jul 2;24(14):3478–3487. doi: 10.1021/bi00335a014. [DOI] [PubMed] [Google Scholar]
  12. Morihara K., Oka T. Subtilisin BPN': inactivation by chloromethyl ketone derivates of peptide substrates. Arch Biochem Biophys. 1970 Jun;138(2):526–531. doi: 10.1016/0003-9861(70)90377-2. [DOI] [PubMed] [Google Scholar]
  13. O'Connell T. P., Malthouse J. P. A study of the stabilization of the oxyanion of tetrahedral adducts by trypsin, chymotrypsin and subtilisin. Biochem J. 1995 Apr 15;307(Pt 2):353–359. doi: 10.1042/bj3070353. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. ONG E. B., SHAW E., SCHOELLMANN G. THE IDENTIFICATION OF THE HISTIDINE RESIDUE AT THE ACTIVE CENTER OF CHYMOTRYPSIN. J Biol Chem. 1965 Feb;240:694–698. [PubMed] [Google Scholar]
  15. Philipp M., Bender M. L. Subtilisin-catalyzed hydrolysis of peptide methyl esters. FEBS Lett. 1974 Jun 15;42(3):282–284. doi: 10.1016/0014-5793(74)80746-5. [DOI] [PubMed] [Google Scholar]
  16. Polgar L., Bender M. L. The reactivity of thiol-subtilisin, an enzyme containing a synthetic functional group. Biochemistry. 1967 Feb;6(2):610–620. doi: 10.1021/bi00854a032. [DOI] [PubMed] [Google Scholar]
  17. Powers J. C., Lively M. O., 3rd, Tippett J. T. Inhibition of subtilisin BPN' with peptide chloromethyl ketones. Biochim Biophys Acta. 1977 Jan 11;480(1):246–261. doi: 10.1016/0005-2744(77)90338-2. [DOI] [PubMed] [Google Scholar]
  18. Primrose W. U., Scott A. I., Mackenzie N. E., Malthouse J. P. A 13C-n.m.r. investigation of ionizations within a trypsin-inhibitor complex. Evidence that the pKa of histidine-57 is raised by interaction with the hemiketal oxyanion. Biochem J. 1985 Nov 1;231(3):677–682. doi: 10.1042/bj2310677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Robillard G., Shulman R. G. High resolution nuclear magnetic resonance studies of the active site of chymotrypsin. I. The hydrogen bonded protons of the "charge relay" system. J Mol Biol. 1974 Jul 5;86(3):519–540. doi: 10.1016/0022-2836(74)90178-8. [DOI] [PubMed] [Google Scholar]
  20. Robillard G., Shulman R. G. High resolution nuclear magnetic resonance study of the histidine--aspartate hydrogen bond in chymotrypsin and chymotrypsinogen. J Mol Biol. 1972 Nov 14;71(2):507–511. doi: 10.1016/0022-2836(72)90366-x. [DOI] [PubMed] [Google Scholar]
  21. Rogers G. A., Bruice T. C. Synthesis and evaluation of a model for the so-called "charge-relay" system of the serine esterases. J Am Chem Soc. 1974 Apr 17;96(8):2473–2481. doi: 10.1021/ja00815a028. [DOI] [PubMed] [Google Scholar]
  22. SCHOELLMANN G., SHAW E. Direct evidence for the presence of histidine in the active center of chymotrypsin. Biochemistry. 1963 Mar-Apr;2:252–255. doi: 10.1021/bi00902a008. [DOI] [PubMed] [Google Scholar]
  23. Shaw E., Ruscica J. The essentiality of histidine in the catalytic action of subtilisin. Covalent modification by a specific reagent. J Biol Chem. 1968 Dec 10;243(23):6312–6313. [PubMed] [Google Scholar]
  24. Shaw E., Springhorn S. Identification of the histidine residue at the active center of trypsin labelled by TLCK. Biochem Biophys Res Commun. 1967 May 5;27(3):391–397. doi: 10.1016/s0006-291x(67)80112-8. [DOI] [PubMed] [Google Scholar]
  25. Tobin J. B., Whitt S. A., Cassidy C. S., Frey P. A. Low-barrier hydrogen bonding in molecular complexes analogous to histidine and aspartate in the catalytic triad of serine proteases. Biochemistry. 1995 May 30;34(21):6919–6924. doi: 10.1021/bi00021a002. [DOI] [PubMed] [Google Scholar]
  26. Warshel A., Naray-Szabo G., Sussman F., Hwang J. K. How do serine proteases really work? Biochemistry. 1989 May 2;28(9):3629–3637. doi: 10.1021/bi00435a001. [DOI] [PubMed] [Google Scholar]
  27. Warshel A., Papazyan A., Kollman P. A. On low-barrier hydrogen bonds and enzyme catalysis. Science. 1995 Jul 7;269(5220):102–106. doi: 10.1126/science.7661987. [DOI] [PubMed] [Google Scholar]
  28. Wright C. S. Comparison of the active site stereochemistry and substrate conformation in -chymotrypsin and subtilisin BPN'. J Mol Biol. 1972 Jun 14;67(1):151–163. doi: 10.1016/0022-2836(72)90391-9. [DOI] [PubMed] [Google Scholar]

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