Zinc is required for the catalytic activity of the human deubiquitinating isopeptidase T.
Two recombinant human isopeptidase T isoforms, ISOT-S and ISOT-L, differing by an insertion of 23 amino acids in ISOT-L, were previously classified as thiol proteases. Both contain one Zn2+-binding site of high-affinity, which is part of a cryptic nitrilo-triacetate-resistant pocket (site 1). A second Zn2+ site (site 2) was disclosed when both isoforms of the holoenzyme were incubated with an excess of Zn2+. The firmly bound Zn2+ of site 1 could be removed either slowly by dialysis against 1,10-phenanthroline at pH 5.5 or rapidly by treatment at pH 3.0 in the presence of 6 M urea followed by gel filtration at neutral pH. Zn2+ in site 1, but not in site 2, is essential for proteolytic activity because apoproteins were inactive. Inhibition of the catalytic activity was not due to a loss of ubiquitin binding capacity. CD spectra of both isoforms disclosed no major structural differences between the apo- and holoenzymes. The reconstitution of apoenzyme with Zn2+ under nondenaturing conditions at pH 5.5 completely restored enzymatic activity, which was indistinguishable from the reconstitution carried out in urea at pH 3.0. Thus, both human ISOTs are either thiol proteases with a local structural Zn2+ or monozinc metalloproteases that might use in catalysis a Zn2+-activated hydroxide ion polarized by Cys335.
Gabriel
Jean-Marc
JM
Department of Medical Biochemistry, University of Geneva, Medical Center, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland. Jean-Marc.Gabriel@medecine.unige.ch
Lacombe
Thierry
T
Carobbio
Stefania
S
Paquet
Nicole
N
Bisig
Ruth
R
Cox
Jos A
JA
Jaton
Jean-Claude
JC
D001051:Apoenzymes / Q000737:chemistry / Q000378:metabolism; D001921:Brain / Q000201:enzymology*; D019759:Carbon-Nitrogen Lyases / Q000187:drug effects* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects*; D002614:Chelating Agents / Q000494:pharmacology*; D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014452:Ubiquitins / Q000378:metabolism; D015032:Zinc / Q000494:pharmacology*
D001051:Apoenzymes; D002614:Chelating Agents; D020035:Holoenzymes; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014452:Ubiquitins; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D015032:Zinc