The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43, with a C-terminal beta-barrel domain.
The crystal structure of an oxidatively stable subtilisin-like alkaline serine protease, KP-43 from Bacillus sp. KSM-KP43, with a C-terminal extension domain, was determined by the multiple isomorphous replacements method with anomalous scattering. The native form was refined to a crystallographic R factor of 0.134 (Rfree of 0.169) at 1.30-A resolution. KP-43 consists of two domains, a subtilisin-like alpha/beta domain and a C-terminal jelly roll beta-barrel domain. The topological architecture of the molecule is similar to that of kexin and furin, which belong to the subtilisin-like proprotein convertases, whereas the amino acid sequence and the binding orientation of the C-terminal beta-barrel domain both differ in each case. Since the C-terminal domains of subtilisin-like proprotein convertases are essential for folding themselves, the domain of KP-43 is also thought to play such a role. KP-43 is known to be an oxidation-resistant protease among the general subtilisin-like proteases. To investigate how KP-43 resists oxidizing reagents, the structure of oxidized KP-43 was also determined and refined to a crystallographic R factor of 0.142 (Rfree of 0.212) at 1.73-A resolution. The structure analysis revealed that Met-256, adjacent to catalytic Ser-255, was oxidized similarly to an equivalent residue in subtilisin BPN'. Although KP-43, as well as proteinase K and subtilisin Carlsberg, lose their hydrolyzing activity against synthetic peptides after oxidation treatment, all of them retain 70-80% activity against proteinaceous substrates. These results, as well as the beta-casein digestion pattern analysis, have indicated that the oxidation of the methionine adjacent to the catalytic serine is not a dominant modification but might alter the substrate specificities.
Nonaka
Tsuyoshi
T
Department of Chemistry, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Fujihashi
Masahiro
M
Kita
Akiko
A
Saeki
Katsuhisa
K
Ito
Susumu
S
Horikoshi
Koki
K
Miki
Kunio
K
D000818:Animals; D001407:Bacillus / Q000201:enzymology; D001665:Binding Sites; D002384:Catalysis; D002417:Cattle; D018360:Crystallography, X-Ray; D004583:Electrons; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000737:chemistry; D005799:Genes, Dominant; D005998:Glycine / Q000031:analogs & derivatives* / Q000737:chemistry; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D010100:Oxygen / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012694:Serine / Q000737:chemistry; D012697:Serine Endopeptidases / Q000737:chemistry*; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry; D013997:Time Factors
D012694:Serine; D008715:Methionine; D012697:Serine Endopeptidases; D013381:Subtilisins; D019286:Endopeptidase K; D010100:Oxygen; D005998:Glycine; C100184:tricine