Characterization of active-site mutants of Schizosaccharomyces pombe phosphoglycerate mutase. Elucidation of the roles of amino acids involved in substrate binding and catalysis.

The roles of a number of amino acids present at the active site of the monomeric phosphoglycerate mutase from the fission yeast Schizosaccharomyces pombe have been explored by site-directed mutagenesis. The amino acids examined could be divided broadly into those presumed from previous related structural studies to be important in the catalytic process (R14, S62 and E93) and those thought to be important in substrate binding (R94, R120 and R121). Most of these residues have not previously been studied by site-directed mutagenesis. All the mutants except R14 were expressed in an engineered null strain of Saccharomyces cerevisiae (S150-gpm:HIS) in good yield. The R14Q mutant was expressed in good yield in the transformed AH22 strain of S. cerevisiae. The S62A mutant was markedly unstable, preventing purification. The various mutants were purified to homogeneity and characterized in terms of kinetic parameters, CD and fluorescence spectra, stability towards denaturation by guanidinium chloride, and stability of phosphorylated enzyme intermediate. In addition, the binding of substrate (3-phosphoglycerate) to wild-type, E93D and R120,121Q enzymes was measured by isothermal titration calorimetry. The results provide evidence for the proposed roles of each of these amino acids in the catalytic cycle and in substrate binding, and will support the current investigation of the structure and dynamics of the enzyme using multidimensional NMR techniques.

Effects of regulatory subunits on the kinetics of protein phosphatase 2A.

Both the scaffold (A) and the regulatory (R) subunits of protein phosphatase 2A regulate enzyme activity and specificity. Heterotrimeric enzymes containing different R-subunits differ in their specific activities for substrates. Kinetic parameters for the dephosphorylation of a phosphopeptide by different oligomeric forms of PP2A were determined to begin to elucidate the molecular basis of regulatory subunit effects on phosphatase activity. Using steady state kinetics and the pH dependence of kinetic parameters, we have explored the effect of the A- and R-subunits on the kinetic and chemical mechanism of PP2A. The regulatory subunits affected a broad range of kinetic parameters. The C-subunit and AC dimer were qualitatively similar with respect to the product inhibition patterns and the pH dependence of kinetic parameters. However, a 22-fold decrease in rate and a 4.7-fold decrease in K(m) can be attributed to the presence of the A-subunit. The presence of the R2alpha (Balpha or PR55alpha) subunit caused an additional decrease in K(m) and changed the kinetic mechanism of peptide dephosphorylation. The R2alpha-subunit also caused significant changes in the pH dependence of kinetic parameters as compared to the free C subunit or AC heterodimer. The data support an important role for the regulatory subunits in determining both the affinity of PP2A heterotrimers for peptide substrates and the mechanism by which they are dephosphorylated.

An anchoring factor targets protein phosphatase 2A to brain microtubules.

Protein phosphatase 2A (PP2A) is a ubiquitously expressed serine/threonine phosphatase composed of a heterodimeric core enzyme that associates with a variety of regulatory subunits. A fraction of brain PP2A associates with microtubules and may play a role in regulating phosphorylation of microtubule-associated proteins. We examined the isoform specificity and the mechanism involved in the association of PP2A with brain microtubules. Only the R2alpha (B/PR55alpha) and R2beta (B/PR55beta) regulatory subunits associated with endogenous neural microtubules. Neither the R2gamma (B/PR55gamma) nor members of the R5 (B'/PR56) family of regulatory subunits co-sedimented with microtubules, although abundant amounts of these proteins were detected in brain. The efficient association of PP2A with microtubules in vitro was dependent on an anchoring activity present in a brain protein fraction containing microtubule-associated and microtubule-interacting proteins. Anchoring factor-dependent association of PP2A with microtubules was specific for the heterotrimeric form of PP2A. The core dimer and the isolated subunits of PP2A had very little affinity for microtubules. Characterization of a fraction enriched in the anchoring factor showed that the activity was a heat labile protein that does not correspond to classical microtubule-associated proteins. The anchoring factor associated with microtubules independently of PP2A. These results indicate the association of PP2A with microtubules can be mediated by an anchoring factor that interacts in an isoform-specific manner with heterotrimeric forms of the phosphatase.

Brain protein serine/threonine phosphatases.

All of the known protein serine/threonine phosphatases are expressed in the brain. These enzymes participate in a variety of signaling pathways that modulate neuronal activity. The multifunctional activity of many serine/threonine phosphatases is achieved through their association with targeting proteins. Identification and analysis of targeting molecules has led to new insights into the functions of protein phosphatases in neuronal signaling. The recent use of transgenic mice has also increased our understanding of the physiological roles of these enzymes in the brain.

Cloning, expression, purification, and characterization of the 6-phosphogluconate dehydrogenase from sheep liver.

The mRNA encoding the 51-kDa subunit of 6-phosphogluconate dehydrogenase (6PGDH) from sheep liver was reverse-transcribed and amplified. The resulting cDNA was reamplified in N-terminal and C-terminal segments and spliced to generate a full-length clone, and an internal cDNA fragment was also amplified. The full-length clone containing the complete coding sequence of the 6PGDH cDNA was sequenced and found to contain two mutations and two deletions in the internal region and two mutations outside of the internal region, an A to G point mutation at position 1407 that resulted in the amino acid change Gln 445 to Arg and a silent mutation at position 1426. The internal clone was sequenced and shown to be free of any mutations; therefore the internal piece was used to replace the same region in the full-length clone to correct the mutations in this region. The mutation at position 1407 which was outside of the internal region was corrected using site-directed mutagenesis. The cDNA with the correct codon was then subcloned into the bacterial expression vector pQE-30 and overproduced in Escherichia coli strain M15. A protein with a subunit molecular weight of 51,000 was expressed at a level of about 4.5% of the total soluble protein in M15 as judged by SDS/PAGE. Cloning into pQE-30 adds six histidines and a short linker to the N-terminus of the enzyme. The recombinant 6PGDH with His-tag was purified using the Ni-NTA affinity column supplied by Qiagen. The purification procedure resulted in a homogeneous protein by SDS/PAGE with 22.4-fold purification with an overall yield of 61%. The recombinant enzyme exhibits kinetic parameters within error identical to those measured for native sheep liver enzyme.

Kinetic and chemical mechanisms of the sheep liver 6-phosphogluconate dehydrogenase.

A complete kinetic characterization of sheep liver 6-phosphogluconate dehydrogenase including product and dead-end inhibition patterns, primary deuterium isotope effects, and the pH dependence of kinetic parameters has been completed in order to determine the kinetic mechanism and obtain information on the chemical mechanism of the enzyme. A rapid equilibrium random kinetic mechanism has been proposed, with product and dead-end inhibition patterns both being symmetric. Ribulose 5-phosphate and 6-sulfogluconate are both competitive with 6-phosphogluconate (6-PG) and noncompetitive with NADP, and NADPH and ATP-ribose are both competitive with NADP and noncompetitive with 6-phosphogluconate. Equal primary deuterium isotope effects of 1.5-2 on DV, DV/KNADP, and DV/K6-PG with 3-deuterio-6-PG confirm a rapid equilibrium random mechanism and show that hydride transfer is at least partially rate limiting in the overall reaction. The maximum velocity is pH dependent, decreasing at low and high pH with slopes of 1 and -1, respectively, and pK values of 6.4 and 8.6. The V/KNADP and V/K6-PG also decrease at low and high pH with slopes of 1 and -1, giving pK values of 6.8 and 8.7 and of 6.9 and 7.8, respectively. The pH rate profiles are consistent with a general acid/general base mechanism where the catalytic residues are involved in binding. Reverse protonation states between the general acid and the general base are proposed where an unprotonated general base accepts a proton from the C-3 hydroxyl of 6-PG concomitant with hydride transfer followed by decarboxylation of the resulting 3-keto intermediate to give an enediol which is protonated by the general acid to form ribulose 5-phosphate. The pH dependence of the pKi profile of the inhibitory analog 5-phosphoribonate decreases at low and high pH with slopes of 1 and -1, respectively, and pKs of 6.2 and 7.4 and suggests that intrinsic pKs are observed in the V/K profiles. The pKs of both the general base and general acid in the E:6-PG complex appears to be perturbed such that the general base decreases from 7.4-7.8 to a value of 6.4-6.8, and the pK of the general acid increases from 6. 2-6.9 to a value of 8.6-8.7, as a result of direct interaction with 6PG. Data are interpreted with regard to the published crystal structures of the E:6-PG, E:NADP, and E:NADPH complexes.