A number of phosphotyrosine-containing peptides derived from the PDGF receptor phosphorylation sites have been synthesised. The peptides were assayed as substrates of the two isoforms (IF1 and IF2) ...of the low
M
r PTPase. The calculated
k
cat,
K
m, and
k
cat/
K
m values indicate that only one peptide is best hydrolysed by IF2 (but not IF1), whose catalytic efficiency averages those previously reported for most PTPases (except the
Yersinia enzyme). This peptide is the only one containing a couple of no bulky hydrophobic residues at the phosphotyrosine
N-side. The determination of the same catalytic parameters in the presence of analogues of the best hydrolysed peptide in which one or both hydrophobic residues were replaced by Asp or Lys residues confirmed the importance of the hydrophobic cluster at the phosphotyrosine
N-side for optimal enzymatic hydrolysis. These findings are discussed in the light of the known IF2 X-ray structure.
An oligonucleotide-directed mutagenesis study was carried out on the five acylphosphatase conserved lysine residues to assess their possible participation in enzyme active site formation and their ...contribution to the enzyme conformational stability. The study was designed to eliminate the ambiguity arising from the presence of a sulfate ion, an enzyme competitive inhibitor, bound to lysine 32 and 68 in the crystal structure of the erythrocyte isoenzyme. Furthermore, previous kinetic studies suggested the presence of residues with pKa=7.9 and 11, tentatively identified as two lysines. The kinetic parameters for the mutants under investigation are not significantly different from those of the wild-type enzyme, demonstrating that none of the lysine residues are involved in catalysis or in substrate binding. In addition, thermal and urea denaturation experiments performed by circular dichroism indicate that the mutated lysine residues do not play a significant role in the enzyme structural stabilization, as the destabilizing energy averages 1.40 kJ/mol. Such results are in agreement with those obtained with other proteins indicating that lysine residues make little contribution to the stability of the native structure.
Enzymatic activity and structure of N-terminus truncated and C-terminus substituted muscle acylphosphatase mutants were investigated by kinetic studies under different conditions and
1H NMR ...spectroscopy, respectively. The N-terminus truncated mutant lacked the first six residues (Δ6), whereas arginine 97 and tyrosine 98 were replaced by glutamine giving two C-terminus substituted mutants (R97Q and Y98Q, respectively). All acylphosphatase forms were obtained by modifications of a synthetic gene coding for the human muscle enzyme which was expressed in
E. coli. The Δ6 deletion mutant elicited a reduced specific activity and a native-like structure. The kinetic and structural properties of R97Q and Y98Q mutants indicate a possible role of Arg-97 in the stabilisation of the active site correct conformation, most likely via back-bone and side chain interactions with Arg-23, the residue involved in phosphate binding by the enzyme. This study also suggests a possible involvement of Tyr-98 in the stabilisation of the acylphosphatase overall structure.
Three mutants of human muscle acylphosphatase in which arginine-23 was replaced by glutamine, histidine and lysine, respectively, were prepared by oligonucleotide-directed mutagenesis of a synthetic ...gene coding for the enzyme. All mutants, purified by affinity chromatography, were almost completely unable to catalyze the hydrolysis of the substrate. 1H-NMR spectroscopy experiments showed the absence of any major conformational changes of the three mutants with respect to the wild-type recombinant enzyme. Equilibrium dialysis experiments demonstrated that the mutated proteins lost the ability of binding inorganic phosphate, a competitive inhibitor of the enzyme. These results strongly support an involvement of arginine-23 at the phosphate binding-site of acylphosphatase, confirming the hypothesis of the existence of a phosphate binding structural motif recently proposed by other authors.
Single crystals of a low molecular weight phosphotyrosine protein phosphatase from bovine liver have been grown. The crystals belong to space group P2
12
12
1, have cell dimensions
a = 46.3 Å,
b - ...62.2 Å,
c = 62.7 Å and diffract to better than 2.0 Å resolution. The crystals are well suited for high resolution X-ray studies.
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