Crystallization and preliminary X-ray analysis of a bacterial psychrophilic enzyme, phosphoglycerate kinase.

The glycolytic enzyme phosphoglycerate kinase (PGK) from the Antarctic microorganism Pseudomonas sp. TACII18 is a cold-adapted enzyme that displays a high specific activity at low temperatures and decreased thermostability relative to its mesophilic counterpart. Herein, the preliminary crystallization and structure solution of psychrophilic PGK in its native form and cocrystallized with 3-phosphoglyceric acid (3-PGA) and the ATP analogue adenylyl imidophosphate (AMP-PNP) is reported. The complexed form of PGK crystallized in 2-3 d at 290 K, whereas the native form of the enzyme required 8-12 months. Morphologically, both crystal forms are similar and X-ray diffraction experiments indicate that the crystals are isomorphous. The crystals diffracted to a resolution of 2.0 A and belong to the space group P3(2). with unit-cell parameters a = b = 58.5, c = 85.4 A.

A study of the K(+)-site mutant of ascorbate peroxidase: mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.

A series of ferric and ferrous derivatives of wild-type ascorbate peroxidase (APX) and of an engineered K(+)-site mutant of APX that has had its potassium cation binding site removed have been examined by electronic absorption and magnetic circular dichroism (MCD) spectroscopy at 4 degrees C. Wild-type ferric APX has spectroscopic properties that are very similar to those of ferric cytochrome c peroxidase (CCP) and likely exists primarily as a five-coordinate high-spin heme ligated on the proximal side by a histidine at pH 7. There is also evidence for minority contributions from six-coordinate high- and low-spin species (histidine-water, histidine-hydroxide, and bis-histidine). The K(+)-site mutant of APX varies considerably in the electronic absorption and MCD spectra in both the ferric and ferrous states when compared with spectra of the wild-type APX. The electronic absorption and MCD spectra of the engineered K(+)-site APX mutant are essentially identical to those of cytochrome b5, a known bis-imidazole (histidine) ligated heme system. It therefore appears that the K(+)-site mutant of APX has undergone a conformational change to yield a bis-histidine coordination structure in both the ferric and ferrous oxidation states at neutral pH. This conformational change is the result of mutagenesis of the protein to remove the K(+)-binding site which is located approximately 8 A from the peroxide binding pocket. Thus, mutations of protein residues on the proximal side of the heme cause changes in iron ligation on the distal side.

Identification of two electron-transfer sites in ascorbate peroxidase using chemical modification, enzyme kinetics, and crystallography.

Chemical and mutagenic modification combined with X-ray crystallography has been used to probe the ascorbate binding site in ascorbate peroxidase (APX). Chemical modification of the single Cys residue in APX with Ellman's reagent (DTNB) blocks the ability of APX to oxidize ascorbate but not other small aromatic phenolic substrates. DTNB-modified APX (APX-TNB) exhibits only 1.3% wild-type activity when ascorbate is used as the substrate but full activity when aromatic substrates, guaiacol or pyrogallol, are used. Stopped-flow studies show that APX-TNB reacts normally with peroxide to give compound I but that the rates of reduction of both compounds I and II by ascorbate are dramatically slowed. Conversion of Cys32 to Ser leads to approximately 70% drop in ascorbate peroxidase activity with no effect on guaiacol peroxidase activity. These results indicate that uncharged aromatic substrates and the anionic ascorbate molecule interact with different sites on APX. The 2.0 A X-ray crystal structure of APX-TNB shows clear electron density for the TNB group covalently attached to Cys32 in all four molecules of the asymmetric unit, indicating complete and specific modification. It appears that the ascorbate site is blocked by DTNB modification which is well removed from the exposed delta-heme edge where aromatic substrates are thought to bind. This is the first experimental evidence indicating that ascorbate oxidation does not occur at the exposed heme edge but at an alternate binding site in the vicinity of Cys32 near Arg172 and the heme propionates.

The role of quaternary interactions on the stability and activity of ascorbate peroxidase.

Point mutations at the dimer interface of the homodimeric enzyme ascorbate peroxidase (APx) were constructed to assess the role of quaternary interactions in the stability and activity of APx. Analysis of the APx crystal structure shows that Glu112 forms a salt bridge with Lys20 and Arg24 of the opposing subunit near the axis of dyad symmetry between the subunits. Two point mutants, E112A and E112K, were made to determine the effects of a neutral (alanine) and repulsive (lysine) mutation on dimerization, stability, and activity. Gel filtration analysis indicated that the ratio of the monomer to dimer increased as the dimer interface interactions went from attractive to repulsive. Differential scanning calorimetry (DSC) data exhibited a decrease in both the transition temperature (Tm) and enthalpy of unfolding (deltaHc) with Tm = 58.3 +/- 0.5 degrees C, 56.0 +/- 0.8 degrees C, and 53.0 +/- 0.9 degrees C and deltaHc = 245 +/- 29 kcal/mol, 199 +/- 38 kcal/mol, and 170 +/- 25 kcal/mol for wild-type APx, E112A, and E112K, respectively. Similar changes were observed based on thermal melting curves obtained by absorption spectroscopy. No change in enzyme activity was found for the E112A mutant, and only a 25% drop in activity was observed for the E112K mutant which demonstrates that the non-Michaelis Menten kinetics of APx is not due to the APx oligomeric structure. The cryogenic crystal structures of the wild-type and mutant proteins show that mutation induced changes are limited to the dimer interface including an alteration in solvent structure.

Spectroscopic characterization of recombinant pea cytosolic ascorbate peroxidase: similarities and differences with cytochrome c peroxidase.

Recombinant pea cytosolic ascorbate peroxidase (APX) has been characterized by resonance Raman (RR) and electronic absorption spectroscopies. The ferric and ferrous forms together with the complexes with fluoride and imidazole have been studied and compared with the corresponding spectra of cytochrome c peroxidase (CCP). Ferric APX at neutral pH is a mixture of 6- and 5-coordinate high-spin and 6-c low-spin hemes, the latter two species being dominant. The results suggest that the low-spin form derives from a water/hydroxo ligand bound to the heme iron and not from a strong internal ligand as observed in CCP at alkaline pH. Two Fe-Im stretching modes are identified, as in CCP, but the RR frequencies confirm a weaker His163-Asp208 hydrogen bond than in CCP, as suggested on the basis of the X-ray structure [Patterson, W. R., and Poulos, T. L. (1995) Biochemistry 34, 4331-4341]. The data show that CCP and APX have markedly different orientations of the vinyl substituents on the heme chromophore resulting from different steric constraints exerted by the protein matrix.

Implementation of universal directly observed therapy at a New York City hospital and evaluation of an out-patient directly observed therapy program.

SETTING: Directly observed therapy (DOT) program for tuberculosis (TB) at a New York City hospital. OBJECTIVE: To describe a specific TB DOT program model utilizing active prospective identification of inpatients, and identify factors associated with patient acceptance of voluntary DOT and with their retention in therapy. METHODS: Recruitment for DOT by daily surveillance of in-patients. DOT was offered as the patient's choice together with concrete services and incentives. On-site DOT was offered in an out-patient clinic. Outreach efforts were initiated when a patient missed one or more DOT visit. RESULTS: During the study period, 95% of 176 in-patients with TB were evaluated for DOT. Of the 137 who were eligible for DOT, 85% (95% confidence interval [CI], 77.5% to 90%) elected to receive DOT. Illicit drug use was independently associated with a higher likelihood of acceptance of DOT (odds ratio[OR], 4.88; 95% CI, 1.5-15.7). Among the 101 patients who received onsite DOT, illicit drug use (OR, 0.21; 95% CI, 0.08-0.6) and previous TB therapy (OR, 0.27; 95% CI, 0.27-0.7) were both independently associated with lower retention in therapy. However, with intensive case management, only 1% of this cohort was lost to follow-up and the overall treatment completion index was 98%. CONCLUSION: In-patient surveillance is a highly effective DOT recruitment strategy. A DOT model which elicits patient participation in discharge plans and offers incentives can yield a high rate of voluntary acceptance. Outpatient case management is a highly effective means of ensuring treatment completion, especially in those at risk for poor retention.

A point mutation of integrin beta 1 subunit blocks binding of alpha 5 beta 1 to fibronectin and invasin but not recruitment to adhesion plaques.

A point mutation in a highly conserved region of the beta 1 subunit, Asp130 to Ala (D130A) substitution, abrogates the Arg-Gly-Asp (RGD)-dependent binding of alpha 5 beta 1 to fibronectin (FN) without disrupting gross structure or heterodimer assembly. The D130A mutation also interferes with binding to invasin, a ligand that lacks RGD sequence. In spite of the lack of detectable FN binding by alpha 5 beta 1(D130A), it was recruited to adhesion plaques formed on FN by endogenous hamster receptors. Thus, intact ligand binding function is not required for recruitment of alpha 5 beta 1 to adhesion plaques. Overexpression of beta 1(D130A) partially interfered with endogenous alpha 5 beta 1 function, thus defining a dominant negative beta 1 integrin mutation.

Modulation of the affinity of integrin alpha IIb beta 3 (GPIIb-IIIa) by the cytoplasmic domain of alpha IIb.

Intracellular signaling alters integrin adhesive functions in inflammation, immune responses, hemostasis, thrombosis, and retinal development. By truncating the cytoplasmic domain of alpha IIb, the affinity of integrin alpha IIb beta 3 for ligand was increased. Reconstitution with the cytoplasmic domain from integrin alpha 5 did not reverse the increased affinity. Thus, the cytoplasmic domain of the alpha subunit of GPIIb-IIIa controls ligand binding affinity, which suggests mechanisms for inside-out transmembrane signaling through integrins. These findings imply the existence of hitherto unappreciated hereditary and acquired thrombotic disorders in humans.