A heat-stable serine proteinase from the extreme thermophilic archaebacterium Sulfolobus solfataricus.
A proteinase was purified to electrophoretic homogeneity from crude extracts of the thermoacidophilic archaebacterium Sulfolobus solfataricus. Molecular mass values assessed by SDS-PAGE and gel filtration were 54 and 118 kDa, respectively, which points to a dimeric structure of the molecule. An isoelectric point of 5.6 was also determined. The enzyme behaved as a chymotrypsin-like serine proteinase, as shown by the inhibitory effects exerted by phenylmethanesulfonyl fluoride, 3,4-dichloroisocoumarin, tosylphenylalaninechloromethyl ketone and chymostatin. Consistently with the inhibition pattern, the enzyme cleaved chromogenic substrates at the carboxyl side of aromatic or bulky aliphatic amino acids; however, it effectively attacked only a small number of such substrates, thus, displaying a specificity much narrower than and clearly different from that of chymotrypsin. This was confirmed by its inability to digest a set of natural substrate proteins, as well as insulin chains A and B; only after alkylation casein was degraded to some extent. Proteinase activity was significantly stimulated by Mn2+ which acted as a mixed-type nonessential activator. The enzyme also displayed a broad pH optimum in the range 6.5-8.0. Furthermore, it was completely stable up to 90 degrees C; above this temperature it underwent first-order thermal inactivation with half-lives ranging from 342 min (92 degrees C) to 7 min (101 degrees C). At 50 degrees C it could withstand 6 M urea and, to some extent, different organic solvents; however, at 95 degrees C it was extensively inactivated by all of these compounds. None of the chemical physical properties of the enzyme, including amino-acid analysis, provided evidence of a possible relation to other well-known microbial serine proteinases.
Burlini
N
N
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Magnani
P
P
Villa
A
A
Macchi
F
F
Tortora
P
P
Guerritore
A
A
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002918:Chymotrypsin / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D006358:Hot Temperature; D008345:Manganese / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology
D000596:Amino Acids; D008345:Manganese; D010664:Phenylmethylsulfonyl Fluoride; D014508:Urea; D012697:Serine Endopeptidases; D002918:Chymotrypsin