title: Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis-Menten mechanism.
abstract: BACKGROUND
  It is known that tandem domains of enzymes can carry out catalysis independently or by collaboration. In the case of cysteine proteases, domain sequestration abolishes catalysis because the active site residues are distributed in both domains. The validity of this argument is tested here by using isolated human ribosomal protein S4, which has been recently identified as an unorthodox cysteine protease.
  
  
  METHODS
  Cleavage of the peptide substrate Z-FR↓-AMC catalyzed by recombinant C-terminal domain of human S4 (CHS4) is studied by fluorescence-monitored steady-state and stopped-flow kinetic methods. Proteolysis and autoproteolysis were analyzed by electrophoresis.
  
  
  RESULTS
  The CHS4 domain comprised of sequence residues 116-263 has been cloned and ovreexpressed in Escherichia coli. The purified domain is enzymatically active. Barring minor differences, steady-state kinetic parameters for catalysis by CHS4 are very similar to those for full-length human S4. Further, stopped-flow transient kinetics of pre-steady-state substrate binding shows that the catalytic mechanism for both full-length S4 and CHS4 obeys the Michaelis-Menten model adequately. Consideration of the evolutionary domain organization of the S4e family of ribosomal proteins indicates that the central domain (residues 94-170) within CHS4 is indispensable.
  
  
  CONCLUSIONS
  The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does.
  
  
  CONCLUSIONS
  Localization of the enzyme function in the C-terminal domain of human S4 provides the only example of a cysteine endoprotease where substrate-mediated intramolecular domain interaction is irrelevant for catalytic activity.
authors.affiliation: School of Chemistry, University of Hyderabad, Hyderabad 500 046, India.
authors.forename: Babu
authors.initials: B
authors.lastname: Sudhamalla
authors.forename: Mahesh
authors.initials: M
authors.lastname: Kumar
authors.forename: Karnati R
authors.initials: KR
authors.lastname: Roy
authors.forename: R Sunil
authors.initials: RS
authors.lastname: Kumar
authors.forename: Abani K
authors.initials: AK
authors.lastname: Bhuyan
mesh_terms: D000595:Amino Acid Sequence; D016923:Cell Death / Q000235:genetics; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism*; D057056:Cysteine Proteases / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics*; D059748:Proteolysis; D012269:Ribosomal Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D012269:Ribosomal Proteins; C031798:ribosomal protein S4; D057056:Cysteine Proteases; D003545:Cysteine
keywords: C-terminal domain of S4; C-terminal domain of human S4; CHS4; Cell growth and proliferation; Human S4X isoform; N-CBZ-Phe-Arg-aminomethylcoumarin; Papain-like cysteine protease; Ribosomal protein S4; Z-FR↓-AMC; rRNA; ribosomal RNA