Cysteine endoprotease activity of human ribosomal protein S4 is entirely due to the C-terminal domain, and is consistent with Michaelis–Menten mechanism Babu Sudhamalla a , Mahesh Kumar b , Karnati R. Roy b , R. Sunil Kumar a , Abani K. Bhuyan a, ⁎ a School of Chemistry, University of Hyderabad, Hyderabad 500 046, India b School of Life Scinces, University of Hyderabad, Hyderabad 500 046, India abstract article info Article history: Received 1 March 2013 Received in revised form 10 May 2013 Accepted 9 June 2013 Available online 19 June 2013 Keywords: C-terminal domain of S4 Ribosomal protein S4 Papain-like cysteine protease Human S4X isoform Cell growth and proliferation Background: It is known that tandem domains of enzymes can carry out catalysis independently or by collabo- ration. 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 pa- rameters 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 indis- pensable. Conclusion: The C-terminal domain can carry out catalysis independently and as efficiently as the full-length human S4 does. Significance: 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. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Numerous recent reports indicate that structural modules or do- mains may participate in the functioning of multidomain enzymes by a variety of manners often unexpected. Domains of bifunctional two-domain enzymes can work sequentially with a defined division of labor to complete a set of activities, but the respective domains may or may not function individually when they are sequestered. For example, the N-terminal domain of the Salmonella virulence factor SrfH performs the non-enzymatic task of vacuole translocation and subcellular targeting within the host, and the C-terminal domain, which is structurally very similar to cysteine proteases, can indepen- dently function as a substrate-specific glutamine deamidase [1]. In another instance, the N- and C-terminal domains of the enzyme FDH (10-formyltetrahydrofolate) dehydrogenase collaborate to execute two sequential reactions. While the N-terminal domain binds folate and functions as a hydrolase to remove the formyl group from the folate substrate, the C-terminal domain acts as a dehydrogenase to oxi- dize the already removed formyl to CO 2 . However, the separately expressed C-terminal domain can independently function as an aldehyde dehydrogenase [2,3]. Similarly, the C-terminal domain of the enzyme ArnA carries out NAD + -dependent decarboxylation of UDP glucuronic acid, and the N-terminal domain performs formyltetrahydrofolate- dependent formylation of UDP-4amino-4 deoxy-L-arabinose. The C-terminal domain separated from the full-length protein shows the NAD + -dependent decarboxylation of UDP glucuronic acid, and its activ- ity is identical to that of the intact enzyme [4]. Reverse gyrase is another case where both interdomain functional collaboration and independent domain function have been reported [5]. Protein tyrosine phosphatase separated from the two tandem SH2 domains at the N-terminus shows several tens of fold higher phosphatase activity than the full-length enzyme [6]. Such structural distinction and independent Biochimica et Biophysica Acta 1830 (2013) 5342–5349 Abbreviations: rRNA, ribosomal RNA; Z-FR↓-AMC, N-CBZ-Phe-Arg-aminomethyl- coumarin; CHS4, C-terminal domain of human S4 ⁎ Corresponding author. Tel.: +91 40 2313 4810. E-mail address: akbsc@uohyd.ernet.in (A.K. Bhuyan). 0304-4165/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bbagen.2013.06.011 Contents lists available at ScienceDirect Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbagen