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

1996 ◽  
Vol 317 (1) ◽  
pp. 35-40 ◽  
Author(s):  
Timothy P. O'CONNELL ◽  
J. Paul G. MALTHOUSE
Keyword(s):  
13C Nmr ◽  
Active Site ◽  
Catalytic Mechanism ◽  
13C Nmr Spectroscopy ◽  
Imidazole Ring ◽  
Histidine Residue ◽  
Ionization State ◽  
Methylene Carbon ◽  
Active Site Histidine

Subtilisin BPN´ has been alkylated using benzyloxycarbonylglycylglycyl[1-13C]phenylalanylchloromethane. Using difference 13C-NMR spectroscopy a single signal due to the 13C-enriched α-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 α-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.

scholarly journals A reinvestigation of residues 64–68 and 175 in papain. Evidence that residues 64 and 175 are asparagine

1970 ◽  
Vol 116 (4) ◽  
pp. 689-692 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Active Site ◽  
Tertiary Structure ◽  
Thiol Group ◽  
Imidazole Ring ◽  
Histidine Residue ◽  
Active Site Cysteine ◽  
Density Map ◽  
A Site ◽  
Electron Density Map ◽  
Active Site Histidine

The tryptophan-containing peptides were isolated from the chymotryptic digest of S-carboxymethylated papain. Residue 175, which is strongly hydrogen-bonded to the active-site histidine residue in the tertiary structure of papain, is asparagine, confirming the work of Kimmel, Rogers & Smith (1965). Its function is probably to maintain the orientation and tautomeric state of the imidazole ring of histidine-159. The amino acid sequence predicted from the electron-density map of papain for residues 64–68 was confirmed, but residue 64 is asparagine, not aspartic acid. This residue, which is about 10 Å from the thiol group of the active-site cysteine-25, cannot therefore be a site of electrostatic attraction for substrates of basic amino acids.

Determination of the preferred tautomeric form of histamine by 13C nmr spectroscopy

1977 ◽  
Vol 55 (5) ◽  
pp. 576-578 ◽  
Author(s):  
William F. Reynolds ◽  
Chau W. Tzeng
Keyword(s):  
Nmr Spectroscopy ◽  
Chemical Shift ◽  
13C Nmr ◽  
Tautomeric Form ◽  
Chemical Shifts ◽  
13C Nmr Spectroscopy ◽  
Imidazole Ring ◽  
Shift Method ◽  
Ph Dependent

The pH-dependent 13C chemical shifts for histamine indicate an approximate 4:1 preference for the Nτ-H tautomer of the imidazole ring, similar to that previously deduced for L-histidine. It is concluded that the 13C chemical shift method is a complimentary technique to the method of determining tautomer preference from pK values. Factors determining the tautomer preference in histamine and L-histidine are discussed.

Natural abundance 13C-NMR spectroscopy for the quantitative determination of fecal fat

2003 ◽  
Vol 36 (7) ◽  
pp. 505-510 ◽  
Author(s):  
P Kunz ◽  
B Künnecke ◽  
I Kunz ◽  
H Lengsfeld ◽  
M von Kienlin
Keyword(s):  
Nmr Spectroscopy ◽  
Quantitative Determination ◽  
13C Nmr ◽  
13C Nmr Spectroscopy ◽  
Natural Abundance ◽  
Fecal Fat

Determination of the structural configuration of cresol–novolak resins by 13C NMR spectroscopy

1982 ◽  
Vol 27 (9) ◽  
pp. 3449-3454 ◽  
Author(s):  
J. A. Carothers ◽  
E. Gipstein ◽  
W. W. Fleming ◽  
T. Tompkins
Keyword(s):  
Nmr Spectroscopy ◽  
13C Nmr ◽  
13C Nmr Spectroscopy ◽  
Structural Configuration

scholarly journals The location of the active-site histidine residue in the primary sequence of papain

1968 ◽  
Vol 108 (5) ◽  
pp. 861-866 ◽  
Author(s):  
S. S. Husain ◽  
G. Lowe
Keyword(s):  
Amino Acid ◽  
Sodium Borohydride ◽  
Active Site ◽  
Iodoacetic Acid ◽  
Cysteine Residue ◽  
Histidine Residue ◽  
Primary Sequence ◽  
Active Site Cysteine ◽  
Active Site Histidine ◽  
Active Site Cysteine Residue

Papain that had been irreversibly inhibited with 1,3-dibromo[2−14C]acetone was reduced with sodium borohydride and carboxymethylated with iodoacetic acid. After digestion with trypsin and α-chymotrypsin the radioactive peptides were purified chromatographically. Their amino acid composition indicated that cysteine-25 and histidine-106 were cross-linked. Since cysteine-25 is known to be the active-site cysteine residue, histidine-106 must be the active-site histidine residue.

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