Substrates and inhibitors of human T-cell leukemia virus type I protease.
HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.
Ding
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YS
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta 30332-0400, USA.
Rich
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Ikeda
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D000595:Amino Acid Sequence; D016282:Aspartic Acid Endopeptidases / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D004789:Enzyme Activation; D017320:HIV Protease Inhibitors / Q000494:pharmacology; D006639:Histidine / Q000235:genetics; D015368:Human T-lymphotropic virus 1 / Q000201:enzymology*; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D021382:Protein Sorting Signals / Q000235:genetics; D011753:Pyrones / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D013379:Substrate Specificity
D017320:HIV Protease Inhibitors; C116428:MES13-099; D009842:Oligopeptides; D011480:Protease Inhibitors; D021382:Protein Sorting Signals; D011753:Pyrones; D011993:Recombinant Fusion Proteins; C066511:JG 365; D006639:Histidine; D016282:Aspartic Acid Endopeptidases; C079711:HTLV-1 protease