Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1 Paolo Paoli a , Alessandra Modesti a , Francesca Magherini a , Tania Gamberi a , Anna Caselli a , Giampaolo Manao a , Giovanni Raugei a,b , Guido Camici a,b, ⁎ , Giampietro Ramponi a,b a Department of Biochemical Sciences, University of Florence, Italy b Center for Research, Transfer and High Education DENOTHE, University of Florence, Italy Received 25 July 2006; received in revised form 13 December 2006; accepted 21 December 2006 Available online 8 January 2007 Abstract We mutated Trp 134 and Tyr 135 of the yeast LMW-PTP to explore their catalytic roles, demonstrating that the mutations of Trp 134 to Tyr or Ala, and Tyr 135 to Ala, all interfere with the formation of the phosphorylenzyme intermediate, a phenomenon that can be seen by the decrease in the kinetic constant of the chemical step (k 3 ). Furthermore, we noted that the Trp 134 to Ala mutation causes a dramatic drop in k cat /K m and a slight enhancement of the dissociation constant K s . The conservative mutant W134Y shows a k cat /K m very close to that of wild type, probably compensating the two-fold decrease of k 3 with an increase in substrate affinity. The Y135A mutation enhances the substrate affinity, but reduces the enzyme phosphorylation rate. The replacement of Trp 134 with alanine interferes with the partition between phosphorylenzyme hydrolysis and phosphotransfer from the phosphorylenzyme to glycerol and abolish the enzyme activation by adenine. Finally, we found that mutation of Trp 134 to Ala causes a dramatic change in the pH-rate profile that becomes similar to that of the D132A mutant, suggesting that an aromatic residue in position 134 is necessary to assist the proper positioning of the proton donor in the transition state of the chemical step. © 2007 Elsevier B.V. All rights reserved. Keywords: PTP; LMW-PTP; Ltp1; Tyrosine phosphorylation; Yeast; Mutagenesis; Aromatic residue 1. Introduction Protein tyrosine phosphatases (PTPs) are signaling enzymes that act in concert with protein tyrosine kinases (PTKs) in regulating several important cellular processes including metabolism, growth, differentiation, transformation, cycle control, and apoptosis [1]. There is accumulating evidence to indicate that the contribution of PTPs to the controlling of the state of protein tyrosine phosphorylation is as relevant as that of PTKs. PTP activity is carefully regulated, and, in most cases, appears to be highly specific. The PTP super-family consists of four main families: the tyrosine specific phospha- tases, the VH1-like dual specificity phosphatases, the Cdc25 phosphatases, and the low molecular weight phosphatases (LMW-PTPs). Despite their extremely limited sequence similarity, all share an active site motif consisting of a cysteine and an arginine separated by five residues (CX 5 R, where C and R are essential residues and X is any amino acid). All PTPs have an identical catalytic mechanism, which involves the formation of a cysteinyl-phosphate covalent intermediate which successively undergoes hydrolysis [2]. LMW-PTP is an enzyme without an extensive sequence homology with other members of the PTP family, but it contains the CX 5 R motif and shares the same catalytic mechanism of other PTPs [3]. Furthermore, the LMW-PTP crystal structure [4] revealed a 3D phosphate binding loop that is structurally identical to that contained in the human placenta PTP1B and Yersinia PTP [5,6]. Genes coding for LMW-PTPs are found in all organisms: they produce enzymes that share a very conserved amino- terminal region containing the P-loop that binds the phosphate Biochimica et Biophysica Acta 1770 (2007) 753 – 762 www.elsevier.com/locate/bbagen Abbreviations: PTP, protein tyrosine phosphatase; PTK, protein tyrosine kinase; LMW, low molecular weight; Ltp1, S. cerevisiae LMW-PTP; pNPP, p-nitrophenyl phosphate; GST, glutathione S-transferase; CD, circular dichroism ⁎ Corresponding author. Dipartimento di Scienze Biochimiche, Viale Morgagni 50, 50134 Firenze, Italy. Tel.: +390554598347; fax: +390554598905. E-mail address: guido.camici@unifi.it (G. Camici). 0304-4165/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2006.12.012