Site-directed mutagenesis of two aromatic residues lining the active site pocket of the yeast Ltp1.
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.
Paoli
Paolo
P
Department of Biochemical Sciences, University of Florence, Italy.
Modesti
Alessandra
A
Magherini
Francesca
F
Gamberi
Tania
T
Caselli
Anna
A
Manao
Giampaolo
G
Raugei
Giovanni
G
Camici
Guido
G
Ramponi
Giampietro
G
D000409:Alanine / Q000378:metabolism; D019943:Amino Acid Substitution; D024322:Amino Acids, Aromatic / Q000737:chemistry / Q000235:genetics*; D001665:Binding Sites; D002384:Catalysis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases / Q000737:chemistry* / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry / Q000235:genetics; D014443:Tyrosine / Q000737:chemistry / Q000235:genetics
D024322:Amino Acids, Aromatic; D029701:Saccharomyces cerevisiae Proteins; D014443:Tyrosine; D014364:Tryptophan; C094972:LTP1 protein, S cerevisiae; D017027:Protein Tyrosine Phosphatases; D000409:Alanine