Abstract:
:1. When ribonuclease T1 [EC 3.1.4.8] (0.125% solution) was treated with a 760-fold molar excess of iodoacetamide at pH 8.0 and 37 degrees, about 90% of the original activity was lost in 24 hr. The half-life of the activity was about 8 hr. The binding ability for 3'-GMP was lost simultaneously. Changes were detected only in histidine and the amino-terminal alanine residues upon amino acid analyses of the inactivated protein and its chymotryptic peptides. The inactivation occurred almost in parallel with the loss of two histidine residues in the enzyme. The pH dependences of the rate of inactivation and that of loss of histidine residues were similar and indicated the implication of a histidine residue or residues with pKa 7.5 to 8 in this reaction. 3'-GMP and guanosine showed some protective effect against loss of activity and of histidine residues. The reactivity of histidine residues was also reduced by prior modification of glutamic acid-58 with iodoacetate, of lysine-41 with maleic or cis-aconitic anhydride or 2,4,6-trinitrobenzenesulfonate or of arginine-77 with ninhydrin. 2. Analyses of the chymotryptic peptides from oxidized samples of the iodoacetamide-inactivated enzyme showed that histidine-92 and histidine-40 reacted with iodoacetamide most rapidly and at similar rates, whereas histidine-27 was least reactive. Alkylation of histidine-92 was markedly slowed down when the Glu58-carboxymethylated enzyme was treated with iodoacetamide. On the other hand, alkylation of histidine-40 was slowed down most in the presence of 3'-GMP. These results suggest that histidine-92 and histidine-40 are involved in the catalytic action, probably forming part of the catalytic site and part of the binding site, respectively, and that histidine-27 is partially buried in the enzyme molecule or interacts strongly with some other residue, thus becoming relatively unreactive.
journal_name
J Biochemjournal_title
Journal of biochemistryauthors
Takahashi Kdoi
10.1093/oxfordjournals.jbchem.a131398keywords:
subject
Has Abstractpub_date
1976-12-01 00:00:00pages
1267-75issue
6eissn
0021-924Xissn
1756-2651journal_volume
80pub_type
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doi:10.1093/oxfordjournals.jbchem.a122425
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:1977-05-01 00:00:00
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a132769
更新日期:1980-02-01 00:00:00
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journal_title:Journal of biochemistry
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更新日期:1988-04-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a121985
更新日期:1987-05-01 00:00:00
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pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a123753
更新日期:1992-03-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:2009-03-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a131833
更新日期:1977-11-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:2004-01-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a135367
更新日期:1985-10-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a135433
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pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:2004-05-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a132803
更新日期:1980-03-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a123982
更新日期:1992-12-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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journal_title:Journal of biochemistry
pub_type: 杂志文章
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更新日期:2006-12-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134833
更新日期:1984-07-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a134853
更新日期:1984-08-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a022539
更新日期:1999-11-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a133555
更新日期:1981-10-01 00:00:00
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journal_title:Journal of biochemistry
pub_type: 杂志文章
doi:10.1093/oxfordjournals.jbchem.a123967
更新日期:1992-12-01 00:00:00