Three-dimensional structure of proteinase K at 0.15-nm resolution.
The crystal and molecular structure of proteinase K was determined by X-ray diffraction data to 0.15-nm resolution. The enzyme belongs to the subtilisin family with an active-site catalytic triad Asp39--His69--Ser224 but is a representative of a subgroup with a free Cys73 close to and 'below' the active His69. Besides this Cys72, proteinase K has two disulfide bonds, Cys34--Cys123 and Cys178--Cys249, which contribute to the stability of the tertiary structure consisting of an extended central parallel beta-sheet decorated by six alpha-helices, three short antiparallel beta-sheets, 18 beta-turns and involving several internal, structurally important water molecules. Proteinase K exhibits two Ca2+-binding sites, one very strong and the other weak, which were the sites of the heavy atoms (Pb2+, Sm3+) used to solve the crystal structure. The weak binding site is liganded to the N and C termini, Thr16 and Asp260, and is only incompletely coordinated by oxygen ligands. The strong binding site is coordinated in the form of a pentagonal bipyramid with the side chain carboxylate of Asp200 and the C = O of Pro175 as apex, and C = O of Val177 and four water molecules in the equatorial plane. Upon removal of this Ca2+, proteinase K loses activity which is interpreted in terms of a local structural deformation involving the substrate-recognition site (Ser132--Gly136), probably associated with a cis----trans isomerization of cis Pro171. Several water molecules are located in the active site. One, W335, is positioned in the 'oxyanion hole' and is displaced by the C = O of the scissile peptide bond of the substrate, as indicated by crystallographic studies with peptide chloromethane inhibitors. Based on these experiments, a reaction mechanism is proposed where the peptide substrate forms a three-stranded antiparallel pleated sheet with the recognition site of proteinase K consisting of Ser132--Leu133--Gly134 on one side and Gly100--Ser101 on the other, followed by expulsion of the oxyanion hole water W335 and hydrolytic cleavage by the Asp39--His69--Serr224 triad. These latter residues display low thermal motion corresponding to well-defined geometry and are hardly accessible to solvent molecules, whereas the recognition-site amino acids are more flexible and partially exposed to solvent.
Betzel
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Institut für Kristallographie, Freie Universität Berlin.
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Saenger
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D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000032:analysis; D002135:Calcium-Binding Proteins / Q000032:analysis; D055598:Chemical Phenomena; D002621:Chemistry; D019286:Endopeptidase K; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis; D011487:Protein Conformation; D012697:Serine Endopeptidases / Q000032:analysis*; D012997:Solvents; D013696:Temperature; D014867:Water / Q000032:analysis; D014961:X-Ray Diffraction
D002135:Calcium-Binding Proteins; D010455:Peptides; D012997:Solvents; D014867:Water; D012697:Serine Endopeptidases; D019286:Endopeptidase K; D002118:Calcium