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P Badola

Publications and source records attributed to P Badola.

4 recordsLinked to original sources
PubMed1997

Escherichia coli diacylglycerol kinase is an evolutionarily optimized membrane enzyme and catalyzes direct phosphoryl transfer.

In this contribution the kinetic mechanism and substrate specificity of Escherichia coli diacylglycerol kinase were examined. Steady state kinetic studies were carried out under mixed micellar conditions using a novel continuous coupled assay system. The kinetic data were consistent with a random equilibrium mechanism, implying that diacylglycerol kinase catalyzes direct phosphoryl transfer from MgATP to diacylglycerol. This was supported by failure to detect an enzyme-phosphate covalent intermediate and by the observation that the bisubstrate analog adenosine 5'-tetraphosphoryl-3-O-(1,2-dihexanoyl)-sn-glycerol inhibits the enzyme (Ki << Km,DAG). While diacylglycerol kinase's kcat/Km is modest compared with the efficiency of many water-soluble enzymes, the enzyme nevertheless appears to be an evolutionarily optimized biocatalyst in the sense that its chemical reaction rate approaches the substrate diffusion-controlled limit. The in vivo rate-limiting step of DAGK's reaction appears to be, in part, the transbilayer diffusion of diacylglycerol from the outer leaflet to the inner leaflet of the cytoplasmic membrane where DAGK's active site is located. DAGK was observed to maintain a high nucleotide substrate specificity, with most of this specificity being expressed in the form of reductions in kcat for ATP analogs.

Adenosine Diphosphate↗
PubMed1997

Escherichia coli diacylglycerol kinase: a case study in the application of solution NMR methods to an integral membrane protein.

Diacylglycerol kinase (DAGK) is a 13-kDa integral membrane protein that spans the lipid bilayer three times and which is active in some micellar systems. In this work DAGK was purified using metal ion chelate chromatography, and its structural properties in micelles and organic solvent mixtures studies were examined, primarily to address the question of whether the structure of DAGK can be determined using solution NMR methods. Cross-linking studies established that DAGK is homotrimeric in decyl maltoside (DM) micelles and mixed micelles. The aggregate detergent-protein molecular mass of DAGK in both octyl glucoside and DM micelles was determined to be in the range of 100-110 kDa-much larger than the sum of the molecular weights of the DAGK trimers and the protein-free micelles. In acidic organic solvent mixtures, DAGK-DM complexes were highly soluble and yielded relatively well-resolved NMR spectra. NMR and circular dichroism studies indicated that in these mixtures the enzyme adopts a kinetically trapped monomeric structure in which it irreversibly binds several detergent molecules and is primarily alpha-helical, but in which its tertiary structure is largely disordered. Although these results provide new information regarding the native oligomeric state of DAGK and the structural properties of complex membrane proteins in micelles and organic solvent mixtures, the results discourage the notion that the structure of DAGK can be readily determined at high resolution with solution NMR methods.

Amino Acid Sequence↗
PubMed1996

Escherichia coli diacylglycerol kinase is an alpha-helical polytopic membrane protein and can spontaneously insert into preformed lipid vesicles.

Escherichia coli diacylglycerol kinase (DAGK) is a 13.2 kDa enzyme which spans the cytoplasmic membrane three times. Functional DAGK was purified to homogeneity using a polyhistidine tag and Ni(II)-chelate chromatography. Transmission Fourier transform infrared spectroscopy (FT-IR) of DAGK in phosphatidylcholine multilayers led to the conclusion that > or = 90 of DAGK's native 121 residues are alpha-helical, consistent with a model in which DAGK consists of two amphipathic alpha-helices and three transmembrane helices. Polarized attenuated total reflection FT-IR studies of DAGK in oriented multilamellae yielded data consistent with a topological arrangement in which the three transmembrane helices are well-aligned with the bilayer normal while the two amphipathic helices are approximately parallel with the membrane plane. The ability of DAGK to spontaneously insert into preformed lipid vesicles was examined using a novel assay system involving DAGK-catalyzed phosphorylation of a fluorescently tagged diacylglycerol. When micellar DAGK is diluted into L alpha-phase vesicles spontaneous insertion of the enzyme is fairly efficient (ca. 30%). DAGK refolding and insertion from delipidated urea-solubilized DAGK into lipid vesicles is also modestly efficient (3.8 +/- 2.1%) above the gel to liquid crystalline phase transition temperature. The insertion studies indicate that the difference in energy barriers (delta delta G++) between pathways leading to catalytically productive folding and insertion of DAGK relative to unproductive pathways is < 4 kcal/mol. However, additional studies carried out with mutant forms of DAGK indicated that the differences between refolding/insertion pathways for DAGK in vivo and in vitro can be significant.

Amino Acid Sequence↗