The structure of the specific phosphate binding loop (P-loop) of bovine protein tyrosine phosphatase (BPTP) is very similar to that present in high M(r) PTPases. Site-directed mutagenesis was used to explore the role of several conserved residues involved in forming the P-loop of BPTP. Thus, Ser-19 and Ser-43 were individually mutated to alanines, and Asn-15 was mutated to alanine and glutamine. The 1H NMR spectra of the mutants showed good conservation of global secondary structure when compared to wild-type enzyme. Kinetic measurements revealed that only S19A and N15A had substantially altered catalytic activities toward p-nitrophenyl phosphate at pH 5.0, with both mutants exhibiting Vmax values that were 0.25-0.33% of wild-type enzyme. Further kinetic analyses of the N15A and S19A mutants were performed using phosphomonoester substrates with varied phenolic leaving groups. For S19A, the slope of the correlation between Vmax and the substrate leaving group pKa was significantly altered, consistent with a change of the rate-determining step from dephosphorylation to phosphorylation. This was confirmed by partitioning experiments employing methanol as an alternative nucleophile in the dephosphorylation step. Thus, mutating Ser-19 to alanine reduced the efficiency of nucleophilic attack by Cys-12. It is concluded that Ser-19 acts to facilitate the ionization and orientation of Cys-12 for optimal reaction as a nucleophile and as a leaving group. It also appears that Asn-15, Ser-19, His-72, and to a lesser extent Ser-43 serve structural functions that allow the active site to adopt an optimal geometry for phosphate binding. The Asn-15 to Ala mutation appears to disrupt the hydrogen-bonding network, with an accompanying alteration of the geometry of the P-loop. These conclusions are also consistent with changes in the stability of the respective proteins, as measured by urea denaturation.

The low molecular weight protein-tyrosine phosphatase (low M(r) PTPase) is an 18-kDa cytoplasmic enzyme of unknown function that has been previously found in several vertebrates. Using an oligonucleotide probe derived from the active site sequence of the mammalian low M(r) PTPases, a Saccharomyces cerevisiae gene that encodes a homolog of this enzyme was cloned by low stringency hybridization. This gene, LTP1, together with a neighboring gene, TKL1, is shown to be located on the right arm of chromosome XVI. The deduced amino acid sequence of its 161-amino acid residue product shows a 39% average identity with that of the mammalian enzymes. The yeast Ltp1 protein was expressed in Escherichia coli, purified to homogeneity, and shown to possess PTPase activity. The recombinant Ltp1 efficiently hydrolyzes phosphotyrosine and a phosphotyrosine-containing peptide, Tyr531-fyn, but it shows low activity toward phosphoserine and phosphothreonine. The catalytic activity of Ltp1 toward a number of substrates was approximately 30-fold lower than the corresponding values measured for the bovine low M(r) PTPase. However, the yeast enzyme was markedly activated by adenine and some purine nucleosides and nucleotides, including cAMP and cGMP. In the case of adenine, the activity of Ltp1 was increased by approximately 30-fold. The high degree of evolutionary conservation of the low M(r) PTPases implies a significant role for this enzyme. However, neither the disruption of the LTP1 gene nor an approximately 10-fold overexpression of its product in S. cerevisiae caused any apparent phenotypic changes under the conditions tested. No proteins related to Ltp1 could be detected in extracts of the ltp1 null mutant, either by immunoblotting or by gel-filtration analysis accompanied by extended kinetic assays, consistent with the conclusion that LTP1 is the only low M(r) PTPase-encoding gene in S. cerevisiae.

Fluorescence steady-state and lifetime measurements have been performed that permit the differentiation of the 2 intrinsic tryptophan residues in bovine low molecular weight phosphotyrosyl protein phosphatase (BPTP). Spectral information was obtained by use of two single-tryptophan mutant proteins, W39F and W49F, and the double mutant protein W39,49F. Fluorescence measurements show that Trp39 is characterized by a large blue shift, a low quantum yield, and a shorter mean lifetime compared to Trp49. Solute fluorescence quenching studies of W39F reveal that Trp49 is highly exposed to the aqueous environment. In contrast, Trp39 is situated within a hydrophobic core and is only partially accessible to quenching agents such as acrylamide, iodide ion, and cesium ion. The fluorescence contributions of Trp39 and Trp49 are additive, and their sum is equivalent to that observed for wild type BPTP. Calculated intramolecular distances between Trp39 or Trp49 and a 5-[[(acetylamino)-ethyl]amino]naphthalene-1- sulfonate group covalently bound at Cys12 or Cys17 of the respective protein mutants, place Trp49 within 10 A and Trp39 at least 20 A from the active site. The fluorescence decay of the single tryptophan mutants and, surprisingly, wild type BPTP were each adequately fitted as biexponentials. The latter is a consequence of the imprecision involved in determining actual minima in a three- and four-exponential fitting. Comparison of quenching results of wild type BPTP with those of the single tryptophan mutant proteins indicates that minor fluorescence components, easily resolved using a biexponential fitting for the mutant proteins, are unresolvable for wild type BPTP. These minor components skewed the weighted magnitudes and induced perturbations in lifetimes for the tryptophan fluorescence of wild type BPTP, which directly influenced the calculated values of Ksv and kq.

The cytochrome c oxidase activity of the bovine heart enzyme decreases substantially at alkaline pH, from 650 s(-1) at pH 7.0 to less than 10 s(-1) at pH 9.75. In contrast, the cytochrome c peroxidase activity of the enzyme shows little or no pH dependence (30-50 s(-1)) at pH values greater than 8.5. Under the conditions employed, it is demonstrated that the dramatic decrease in oxidase activity at pH 9.75 is fully reversible and not due to a major alkaline-induced conformational change in the enzyme. Furthermore, the Km values for cytochrome c interaction with the enzyme were also not significantly different at pH 7.8 and pH 9.75, suggesting that the pH dependence of the activity is not due to an altered interaction with cytochrome c at alkaline pH. However, at alkaline pH, the steady-state reduction level of the hemes increased, consistent with a slower rate of electron transfer from heme a to heme a3 at alkaline pH. Since it is well established that the rate of electron transfer from heme a to heme a3 is proton-coupled, it is reasonable to postulate that at alkaline pH, proton uptake becomes rate-limiting. The fact that this is not observed when hydrogen peroxide is used as a substrate in place of O2 suggests that the rate-limiting step is proton uptake via the K-channel associated with the reduction of the heme a3/CuB center prior to the reaction with O2. This step is not required for the reaction with H2O2, as shown previously in the examination of mutants of bacterial oxidases in which the K-channel was blocked. It is concluded that at pH values near 10, the delivery of protons via the K-channel becomes the rate-limiting step in the catalytic cycle with O2, so that the behavior of the bovine enzyme resembles that of the K-channel mutants in the bacterial enzymes.

The low-resolution three-dimensional structure of purified native beef heart mitochondrial cytochrome c oxidase (COX) in asolectin unilamellar liposomes has been measured by small-angle neutron scattering under the conditions where the protein remains fully functional. From a neutron scattering perspective, the use of mixed-lipid liposomes provided for a more homogeneous matrix than can be achieved using a single lipid. As a result, the measurements were able to be performed under conditions where the liposome scattering was essentially eliminated (contrast-matched conditions). The protein structure in the membrane was modeled as a simple parallelepiped with side lengths of (59 × 70 × 120) Å with uncertainties, respectively, (11, 12, 20 Å). The molecular mass calculated for a typical protein with this volume is estimated to be (410 ± 124) kDa, which indicates the mass of a COX dimer. The longest dimension has some uncertainty due to intermolecular scattering contributing to the data. Nevertheless, that length was estimated using an average protein density and the known dimer molecular mass. Using the same cross sectional dimensions for the structure, the length is estimated to be 120 Å. However, the measured scattering curve of the dimer in the liposome differs significantly from that calculated from the X-ray structure of the dimer in a crystal of mixed micelles (PDB 3AG1). The calculated SANS scattering from the crystal structure was fit with a parallelepiped, measuring (59 × 101 × 129) Å with fitting uncertainties, respectively, (2, 3, 3 Å). Our results suggest that COX is a functional dimer when reconstituted into mixed-lipid liposomes.