No abstract

Two-component signaling systems are used by bacteria, plants, and lower eukaryotes to adapt to environmental changes. The first component, a protein kinase, responds to a signal by phosphorylating the second component; a response regulator protein that often acts by inducing the expression of specific genes. Response regulators also have an autophosphatase activity that ensures that the proteins are not permanently activated by phosphorylation. The magnitude of this activity varies by at least 1000-fold between various response regulators, and the molecular features responsible for this varied autophosphatase activity have not been clearly defined. Using wild-type and mutant derivatives of the sporulation response regulator Spo0F, it has been demonstrated that a key residue in determining the magnitude of this activity is that at position 56 of Spo0F approximately P; this residue is adjacent to the site of phosphorylation, Asp 54. For example, Spo0F approximately P K56N has a 23-fold greater autophosphatase activity (t1/2 = 8 min) than wild-type Spo0F approximately P (t1/2 = 180 min). It is suggested that, by analogy to the GTPase activity of p21(ras) and by examining the crystallographic structure of Spo0F, that the carboxyamide of the mutant Asn 56 may favorably position a catalytic water near the protein acyl phosphate to promote Spo0F approximately P K56N hydrolysis. It is also deduced that Lys 56 in the wild-type protein is critical for the efficient interaction and phosphoryl transfer between Spo0F and it's cognate protein kinase, KinA. Comparison of the known response regulators shows that inefficient autophosphatases (t1/2 on the order of hours) typically contain an amino acid residue with a long side chain at the position equivalent to 56 in Spo0F, whereas efficient autophosphatases (t1/2 on the order of minutes) frequently contain a residue with a carboxyamide or carboxylate side chain at this position. It appears that, by altering residues adjacent to the active site, the autophosphatase activity of response regulator proteins has been attenuated to match the diverse biological roles played by these proteins.

Spo0F is a secondary messenger in the "two-component" system controlling the sporulation of Bacillus subtilis. Spo0F, like the chemotaxis protein CheY, is a single-domain protein homologous to the N-terminal activator domain of the response regulators. We recently reported the crystal structure of a phosphatase-resistant mutant Y13S of Spo0F with Ca2+ bound in the active site. The crystal structure of wild-type Spo0F in the absence of a metal ion is presented here. A comparison of the two structures reveals that the cation induces significant changes in the active site. In the present wild-type structure, the carboxylate of Asp11 points away from the center of the active site, whereas when coordinated to the Ca2+, as in the earlier structure, it points toward the active site. In addition, Asp54, the site of phosphorylation, is blocked by a salt bridge interaction of an Arg side chain from a neighboring molecule. From fluorescence quenching studies with Spo0F Y13W, we found that only the amino acid Arg binds to Spo0F in a saturable manner (Kd = 15 mM). This observation suggests that a small molecule with a shape complementary to the active site and having a guanidinium group might inhibit phosphotransfer between response regulators and their cognate histidine kinases.

An aqueous two phase system of polyethylene glycol (PEG) and salts was evaluated for separation and purification of alkaline proteases from chicken intestine. Among the different salts evaluated potassium phosphate and sodium citrate gave higher enzyme yield (73.5% and 69.7% respectively) and enzyme purification (5.3 and 7.4 fold) in PEG rich upper phase. Increase in concentration of sodium citrate in the system resulted in reduction in enzyme yield and enzyme purification factor, with 15% salt showing highest enzyme yield (59.8%) and purification (6.7 fold). Initial protein concentration in the system did not show any specific trend on the partition behavior of the enzyme. The temperature at which the system is incubated did not show any significant (p ≥ 0.05) effect on enzyme partition and purification. Increasing the PEG concentration in the system from 15 to 25% resulted in reduction in enzyme yield from 53.7 to 21.9% and enzyme purification from 5 fold to 1.4 fold in PEG rich upper phase. pH also had a significant (p < 0.05) effect on the partition of the enzyme to the upper phase with highest purification (3.4 fold) at pH 9.0 and higher enzyme yield (46.2%) at pH 10.