Effect of temperature and pressure on the proteolytic specificity of the recombinant 20S proteasome from Methanococcus jannaschii.
The hydrolytic specificity of the recombinant 20S proteasome from the deep-sea thermophile Methanococcus jannaschii was evaluated toward oxidized insulin B-chain across a range of temperatures (35 degrees, 55 degrees, 75 degrees, and 90 degrees C) and hydrostatic pressures (1, 250, 500, and 1,000 atm). Of the four temperatures considered, the same maximum overall hydrolysis rate was observed at both 55 degrees and 75 degrees C, which are much lower than the T(opt) of 116 degrees C previously observed for a small amide substrate (Michels and Clark 1997). At 35 degrees C the rates of cleavage were highest at the carboxyl side of glutamine and leucine, whereas at the three higher temperatures, the most rapid cleavages occurred after leucine and glutamic acid residues. The distribution of proteolytic fragments and the cleavage sequence also varied between the lowest and higher temperatures. Application of hydrostatic pressure did not increase proteasome activity, as observed previously for the amide substrate (Michels and Clark 1997), but instead significantly reduced the overall conversion of the polypeptide substrate. Overall cleavage patterns observed for the recombinant M. jannaschii proteasome were similar to those reported previously for Thermoplasma acidophilum (Akopian et al. 1997) and human proteasomes (Dick et al. 1991), indicating that proteasome specificity has been conserved despite significant environmental diversity.
Frankenberg
Rob J
RJ
Bayer Health Care, Berkeley, California, USA.
Andersson
Maria
M
Clark
Douglas S
DS
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007328:Insulin / Q000378:metabolism; D013058:Mass Spectrometry; D017017:Methanococcus / Q000201:enzymology*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000737:chemistry / Q000378:metabolism*; D011312:Pressure; D046988:Proteasome Endopeptidase Complex; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
D007328:Insulin; D009097:Multienzyme Complexes; D011994:Recombinant Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex