[Serine proteinase from the archaebacterium Halobacterium mediterranei--an analog of eubacterium subtilisin].
A homogeneous serine proteinase was isolated from cultural filtrates of the extreme halophilic bacteria Halobacterium mediterranei 1538 using affinity chromatography on bacitracin-Sepharose, ultrafiltration and gel filtration on Sephadex G-75, with a 48% yield and 260-fold purification. The enzyme was completely inactivated by specific inhibitors of serine proteinases, PMSF and DFP, as well as by Hg2+ and PCMB. The enzyme activity was strongly dependent of NaCl concentration, the enzyme being inactivated below 0.75 M NaCl. Inactivation of the enzyme was also seen in the presence of 2-7% organic solvents. The pH optimum for Glp-Ala-Ala-Leu-pNA hydrolysis is 8.0-8.5; Km is 0.14 mM, kcat is 36.9 s-1. The stability optimum lies at pH 5.5-8.0, temperature optimum is at 55 degrees C. The enzyme molecular weight is 41,000 Da; pI is 7.5. The substrate specificity of the enzyme is comparable to that of secretory subtilisins; the extent of protein substrate hydrolysis is similar to that of proteinase K. The N-terminal sequence of Halobacterium mediterranei serine proteinase, Asp-Thr-Ala-Asn-Asp-Pro-Lys-Tyr-Gly-Ser-Gln-Tyr-Ala-Pro-Gln-Lys-Val-Asn- Ala- Asp-, reveals a 50% homology with the aminoterminal sequence of Thermoactinomyces vulgaris serine proteinase. Hence, the serine proteinase secreted by halophilic bacteria may be considered as a structural and functional analog of eubacterial enzymes.
Rudenskaia
G N
GN
Revina
L P
LP
Griaznova
Iu B
IuB
Lysogorskaia
E N
EN
Oksenoĭt
E S
ES
Filippova
I Iu
IIu
Stepanov
V M
VM
Ivanova
I I
II
D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology*; D002850:Chromatography, Gel; D004795:Enzyme Stability; D006217:Halobacterium / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013379:Substrate Specificity; D013381:Subtilisins / Q000235:genetics / Q000378:metabolism*
D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases; D013381:Subtilisins