The three-dimensional structure of Asn102 mutant of trypsin: role of Asp102 in serine protease catalysis.

The structure of the Asn102 mutant of trypsin was determined in order to distinguish whether the reduced activity of the mutant at neutral pH results from an altered active site conformation or from an inability to stabilize a positive charge on the active site histidine. The active site structure of the Asn102 mutant of trypsin is identical to the native enzyme with respect to the specificity pocket, the oxyanion hole, and the orientation of the nucleophilic serine. The observed decrease in rate results from the loss of nucleophilicity of the active site serine. This decreased nucleophilicity may result from stabilization of a His57 tautomer that is unable to accept the serine hydroxyl proton.

[1]  W. Rutter,et al.  The catalytic role of the active site aspartic acid in serine proteases. , 1987, Science.

[2]  W. Bachovchin.  15N NMR spectroscopy of hydrogen-bonding interactions in the active site of serine proteases: evidence for a moving histidine mechanism. , 1986, Biochemistry.

[3]  N. Xuong,et al.  Software for a diffractometer with multiwire area detector. , 1985, Methods in enzymology.

[4]  W. Rutter,et al.  Structure of two related rat pancreatic trypsin genes. , 1984, The Journal of biological chemistry.

[5]  A. Kossiakoff,et al.  Direct determination of the protonation states of aspartic acid-102 and histidine-57 in the tetrahedral intermediate of the serine proteases: neutron structure of trypsin. , 1981, Biochemistry.

[6]  L. Delbaere,et al.  Structures of product and inhibitor complexes of Streptomyces griseus protease A at 1.8 A resolution. A model for serine protease catalysis. , 1980, Journal of molecular biology.

[7]  Robert M. Stroud,et al.  The accuracy of refined protein structures: comparison of two independently refined models of bovine trypsin , 1978 .

[8]  N. Xuong,et al.  The electron stationary picture method for high-speed measurement of reflection intensities from crystals with large unit cells , 1978 .

[9]  P. W. Tucker,et al.  Mechanism of charge compensation and impairment of co-operative functions in haemoglobin Tacoma (Arg B12(30)β → ser) , 1977 .

[10]  R. Stroud,et al.  Difference Fourier refinement of the structure of DIP‐trypsin at 1.5 Å with a minicomputer technique , 1977 .

[11]  R. Huber,et al.  Crystal structure of bovine trypsinogen at 1-8 A resolution. II. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin. , 1977, Journal of molecular biology.

[12]  R. Stroud,et al.  Structure of bovine trypsinogen at 1.9 A resolution. , 1977, Biochemistry.

[13]  R M Stroud,et al.  pH dependence of tritium exchange with the C-2 protons of the histidines in bovine trypsin. , 1976, Biochemistry.

[14]  N. W. Isaacs,et al.  A method for fitting satisfactory models to sets of atomic positions in protein structure refinements , 1976 .

[15]  W. Bode,et al.  The refined crystal structure of bovine beta-trypsin at 1.8 A resolution. II. Crystallographic refinement, calcium binding site, benzamidine binding site and active site at pH 7.0. , 1975, Journal of molecular biology.

[16]  G. G. Christoph,et al.  Silver ion inhibition of serine proteases: crystallographic study of silver-trypsin. , 1974, Biochemical and biophysical research communications.

[17]  R. Stroud,et al.  Structure and specific binding of trypsin: comparison of inhibited derivatives and a model for substrate binding. , 1974, Journal of molecular biology.

[18]  A. Fersht,et al.  The charge relay system in chymotrypsin and chymotrypsinogen. , 1973, Journal of molecular biology.

[19]  H. Neurath,et al.  Inactivation of bovine trypsinogen and chymotrypsinogen by diisopropylphosphorofluoridate. , 1972, Proceedings of the National Academy of Sciences of the United States of America.

[20]  R. Henderson.  Structure of crystalline alpha-chymotrypsin. IV. The structure of indoleacryloyl-alpha-chyotrypsin and its relevance to the hydrolytic mechanism of the enzyme. , 1970, Journal of molecular biology.

[21]  D. Blow,et al.  Role of a Buried Acid Group in the Mechanism of Action of Chymotrypsin , 1969, Nature.

[22]  H. Neurath,et al.  Peptides combined with 14C-diisopropyl phosphoryl following degradation of 14C-DIP-trypsin with alpha-chymotrypsin. , 1956, Biochimica et biophysica acta.