Biomedical subjects

M F Roberts

Publications and source records attributed to M F Roberts.

At least 19 recordsLinked to original sources

PubMed2001

Binding of proteolytically processed phospholipase D from Streptomyces chromofuscus to phosphatidylcholine membranes facilitates vesicle aggregation and fusion.

Ca(2+)-dependent phospholipase D is secreted from Streptomyces chromofuscus as an intact enzyme of 57 kDa (PLD(57)). Under certain growth conditions, PLD is proteolytically cleaved and activated to form PLD(42/20) (named for the apparent size of the peptides). The PLD(42) catalytic core and 20 kDa C-terminal domain remain tightly associated through noncovalent interactions. In the presence of Ba(2+) (to enhance protein binding to zwitterionic vesicles without hydrolysis of substrate), PLD(42/20), but not PLD(57), induces POPC vesicle leakiness as measured by entrapped CF leakage. PLD(42/20) also induces vesicle fusion (as measured by light scattering, fluorescence quenching, and cryo-TEM) under these conditions (1 mM POPC, 5 mM Ba(2+)); neither PLD(42) nor PLD(20) alone can act as a fusogen. For intact PLD(57) to cause CF leakiness, the soluble activator diC(4)PA must be present. However, even with diC(4)PA, PLD(57) does not induce significant vesicle fusion. In the absence of metal ions, all PLD forms bind to PC vesicles doped with 10 mol % PA. Again, only PLD(42/20) is fusogenic and causes aggregation and fusion on a rapid time scale. Taken together, these data suggest that activated PLD(42/20) inserts more readily into the lipid bilayer than other PLD forms and creates structures that allow bilayers to fuse. Cleavage of the PLD(57) by a secreted protease to generate PLD(42/20) occurs in the late stages of S. chromofuscus cell cultures. Production of this more active and fusogenic enzyme may play a role in nutrient scavenging in stationary phase cultures.

4-Chloro-7-nitrobenzofurazan↗

PubMed2001

Switching osmolyte strategies: response of Methanococcus thermolithotrophicus to changes in external NaCl.

Methanococcus thermolithotrophicus, a thermophilic methanogenic archaeon, produces and accumulates beta-glutamate and L-alpha-glutamate as osmolytes when grown in media with <1 M NaCl. When the organism is adapted to grow in >1 M NaCl, a new zwitterionic solute, N(epsilon)-acetyl-beta-lysine, is synthesized and becomes the dominant osmolyte. Several techniques, including in vivo and in vitro NMR spectroscopy, HPLC analyses of ethanol extracts, and potassium atomic absorption, have been used to monitor the immediate response of M. thermolithotrophicus to osmotic stress. There is a temporal hierarchy in the response of intracellular osmolytes. Changes in intracellular K(+) occur within the first few minutes of altering the external NaCl. Upon hypoosmotic shock, K(+) is released from the cell; relatively small changes occur in the organic osmolyte pool on a longer time scale. Upon hyperosmotic shock, M. thermolithotrophicus immediately internalizes K(+), far more than would be needed stoichiometrically to balance the new salt concentration. This is followed by a decrease to a new K(+) concentration (over 10-15 min), at which point synthesis and accumulation of primarily L-alpha-glutamate occur. Once growth of the M. thermolithotrophicus culture begins, typically 30-100 min after the hyperosmotic shock, the intracellular levels of organic anions decrease and the zwitterion (N(epsilon)-acetyl-beta-lysine) begins to represent a larger fraction of the intracellular pool. The observation that N(epsilon)-acetyl-beta-lysine accumulation occurs in osmoadapted cells but not immediately after osmotic shock is consistent with the hypothesis that lysine 2,3-aminomutase, an enzyme involved in N(epsilon)-acetyl-beta-lysine synthesis, is either not present at high levels or has low activity in cells grown and adapted to lower NaCl. That lysine aminomutase specific activity is 8-fold lower in protein extracts from cells adapted to low NaCl compared to those adapted to 1.4 M NaCl supports this hypothesis.

Chromatography, High Pressure Liquid↗

PubMed2001

Crystal structure and catalytic mechanism of the MJ0109 gene product: a bifunctional enzyme with inositol monophosphatase and fructose 1,6-bisphosphatase activities.

Inositol monophosphatase (EC 3.1.3.25) in hyperthermophilic archaea is thought to play a role in the biosynthesis of di-myo-inositol-1,1'-phosphate (DIP), an osmolyte unique to hyperthermophiles. The Methanococcus jannaschii MJ109 gene product, the sequence of which is substantially homologous to that of human inositol monophosphatase, exhibits inositol monophosphatase activity but with substrate specificity that is broader than those of bacterial and eukaryotic inositol monophosphatases (it can also act as a fructose bisphosphatase). To understand its substrate specificity as well as the poor inhibition by Li(+) (a potent inhibitor of the mammalian enzyme), we have crystallized the enzyme and determined its three-dimensional structure. The overall fold, as expected, is similar to that of the mammalian enzyme, but the details suggest a closer relationship to fructose 1,6-bisphosphatases. Three complexes of the MJ0109 protein with substrate and/or product and inhibitory as well as activating metal ions suggest that the phosphatase mechanism is a three-metal ion assisted catalysis which is in variance with that proposed previously for the human inositol monophosphatase.

Amino Acid Sequence↗

PubMed2001

beta-Glutamate as a substrate for glutamine synthetase.

The conversion of beta-glutamate to beta-glutamine by archaeal and bacterial glutamine synthetase (GS) enzymes has been examined. The GS from Methanohalophilus portucalensis (which was partially purified) is capable of catalyzing the amidation of this substrate with a rate sevenfold less than the rate obtained with alpha-glutamate. Recombinant GS from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus were considerably more selective for alpha-glutamate than beta-glutamate as a substrate. All the archaeal enzymes were much less selective than the two bacterial GS (from Escherichia coli and Bacillus subtilis), whose specific activities towards beta-glutamate were much smaller than rates with the alpha-isomer. These results are discussed in light of the observation that beta-glutamate is accumulated as an osmolyte in many archaea while beta-glutamine (produced by glutamine synthetase) is used as an osmolyte only in M. portucalensis.

Animals↗

PubMed2001

An evaluation of the first year's experience with a low-cost telemedicine link in Bangladesh.

In July 1999, the Swinfen Charitable Trust in the UK established a telemedicine link in Bangladesh, between the Centre for the Rehabilitation of the Paralysed (CRP) in Dhaka and medical consultants abroad. This low-cost telemedicine system used a digital camera to capture still images, which were then transmitted by email. During the first 12 months, 27 telemedicine referrals were made. The following specialties were consulted: neurology (44%), orthopaedics (40%), rheumatology (8%), nephrology (4%) and paediatrics (4%). Initial email replies were received at the CRP within a day of referral in 70% of cases and within thee days in 100%, which shows that store-and-forward telemedicine can be both fast and reliable. Telemedicine consultation was complete within three days in 14 cases (52%) and within three weeks in 24 cases (89%). Referral was judged to be beneficial in 24 cases (89%), the benefits including establishment of the diagnosis, the provision of reassurance to the patient and referring doctor, and a change of management. Four patients (15% of the total) and their families were spared the considerable expense and unnecessary stress of travelling abroad for a second opinion, and the savings from this alone outweighed the set-up and running costs in Bangladesh. The latter are limited to an email account with an Internet service provider and the local-rate telephone call charges from the CRP. This successful telemedicine system is a model for further telemedicine projects in the developing world.

Adolescent↗

PubMed2000

Inositol-1-phosphate synthase from Archaeoglobus fulgidus is a class II aldolase.

A gene putatively identified as the Archaeoglobus fulgidus inositol-1-phosphate synthase (IPS) gene was overexpressed to high level (about 30-40% of total soluble cellular proteins) in Escherichia coli. The recombinant protein was purified to homogeneity by heat treatment followed by two column chromatographic steps. The native enzyme was a tetramer of 168 +/- 4 kDa (subunit molecular mass of 44 kDa). At 90 degrees C the K(m) values for glucose-6-phosphate and NAD(+) were estimated as 0.12 +/- 0.04 mM and 5.1 +/- 0.9 microM, respectively. Use of (D)-[5-(13)C]glucose-6-phosphate as a substrate confirmed that the stereochemistry of the product of the IPS reaction was L-myo-inositol-1-phosphate. This archaeal enzyme, with the highest activity at its optimum growth temperature among all IPS reported (k(cat) = 9.6 +/- 0.4 s(-1) with an estimated activation energy of 69 kJ/mol), was extremely heat stable. However, the most unique feature of A. fulgidus IPS was that it absolutely required divalent metal ions for activity. Zn(2+) and Mn(2+) were the best activators with K(D) approximately 1 microM, while NH(4)(+) (a critical activator for all the other characterized IPS enzymes) had no effect on the enzyme. These properties suggested that this archaeal IPS was a class II aldolase. In support of this, stoichiometric reduction of NAD(+) to NADH could be followed spectrophotometrically when EDTA was present along with glucose-6-phosphate.

Aldehyde-Lyases↗

PubMed2000

Osmoadaptation and osmoregulation in archaea.

The response of archaea to changes in external NaCl is reviewed and compared to what is known about osmoadaptation and osmoregulation in bacteria and eukaryotes. Cells placed in altered external NaCl exhibit short term and long term responses. The earliest events are likely to be water movement through aquaporin-like channels (efflux if external NaCl has been increased, influx into the cell if the external NaCl has been decreased) and ion movement (e.g., K+ moving in the direction opposite to water flow) through channels sensitive to osmotic pressure. Accumulation of organic solutes, either by uptake from the medium or de novo synthesis, is triggered after these initial changes. Archaea have some unique organic solutes (osmolytes) that are not used by other organisms. These as well as other more common solutes have a role in stabilizing macromolecules from denaturation. Many osmolytes are distinguished by their stability in the cell and their lack of strong interactions with cellular components. A cell may respond by accumulating one or more temporary osmolytes, then over time readjust the intracellular solute distribution to what is optimal for cell growth under the new conditions. Coupled with the movement and accumulation of solutes is the induction of stress proteins (e.g., chaperonins) and, in some cases, transcriptional regulation of key enzymes. The response to NaCl stress of Methanococcus thermolithotrophicus is presented as an example of how one particular archaeon responds and adapts to altered osmotic pressure. Clearly, the detailed response of other archaea to osmotic stress will be needed in order to identify features (aside from some of the organic osmolytes) unique to the organisms in this kingdom.

Adaptation, Physiological↗

PubMed2000

Overexpression, purification, and analysis of complementation behavior of E. coli SuhB protein: comparison with bacterial and archaeal inositol monophosphatases.

The E. coli suhB gene product, which has been suggested to participate in posttranscriptional control of gene expression, also possesses inositol-1-phosphatase (I-1-Pase) activity. To test if SuhB I-1-Pase activity is sufficient for its function in cells, we have cloned the genes for three other I-1-Pases (from the archaea Methanococcus jannaschii and Archaeoglobus fulgidus, and from the bacterium Thermotoga maritima) into the E. coli expression vector pET23a(+) and examined if these extragenic I-1-Pases could complement the suhB mutation in E. coli strain CG1307 (which also has a mutation in dnaB and a cold-sensitive phenotype). None of these I-1-Pase genes restored growth at 30 degrees C although they generated active I-1-Pase enzymes (as measured by I-1-Pase specific activities of crude protein extracts from the transformed CG1307 cells). In contrast, the pET23a(+) recombinant plasmid with the wild-type E. coli suhB gene complemented the cold sensitivity of the chromosomal mutant suhB and restored the temperature-sensitive growth of the dnaB mutation in the double mutant strain CG1307. Further evidence that this relief of the suppressor behavior of the suhB mutation is not related to the I-1-Pase activity of the SuhB protein was provided by construction of the E. coli SuhB mutant D87N. This mutant protein is inactive as an I-1-Pase but fully functional in changing the temperature sensitivity of the E. coli double mutant strain. Therefore, I-1-P phosphatase activity is neither sufficient nor required for complementation of suhB mutant suppressor effects. The wild-type E. coli SuhB protein was also overexpressed to very high levels and purified to homogeneity in high yield (1 mg/10 mL of culture). The major differences of the E. coli I-1-Pase from all the other characterized I-1-Pases are that it exists as a monomer (rather than a dimer or tetramer) in solution and is more hydrophobic. These physical differences, rather than the I-1-Pase activity, may be involved in the biological role of wild-type SuhB in E. coli.

Amino Acid Sequence↗

PubMed2000

MJ0109 is an enzyme that is both an inositol monophosphatase and the 'missing' archaeal fructose-1,6-bisphosphatase.

In sequenced genomes, protein coding regions with unassigned function constitute between 10 and 50% of all open reading frames. Often key enzymes cannot be identified using sequence homology searches. For example, despite the fact that methanogens have an apparently functional gluconeogenesis pathway, standard tools have been unable to identify a fructose-1,6-bisphosphatase (FBPase) gene in the sequenced Methanoccocus jannaschii genome. Using a combination of functional and structural tools, we have shown that the protein product of the M. jannaschii gene MJ0109, which had been tentatively annotated as an inositol monophosphatase (IMPase), has both IMPase and FBPase activities. Moreover, several gene products annotated as IMPases from different thermophilic organisms also possess FBPase activity. Thus, we have found the FBPase that was 'missing' in thermophiles and shown that it also functions as an IMPase.

Amino Acid Sequence↗

PubMed1999

The role of interfacial binding in the activation of Streptomyces chromofuscus phospholipase D by phosphatidic acid.

The Streptomyces chromofuscus phospholipase D (PLD) cleavage of phosphatidylcholine in bilayers can be enhanced by the addition of the product phosphatidic acid (PA). Other anionic lipids such as phosphatidylinositol, oleic acid, or phosphatidylmethanol do not activate this PLD. This allosteric activation by PA could involve a conformational change in the enzyme that alters PLD binding to phospholipid surfaces. To test this, the binding of intact PLD and proteolytically cleaved isoforms to styrene divinylbenzene beads coated with a phospholipid monolayer and to unilamellar vesicles was examined. The results indicate that intact PLD has a very high affinity for PA bilayers at pH >/= 7 in the presence of EGTA that is weakened as Ca(2+) or Ba(2+) are added to the system. Proteolytically clipped PLD also binds tightly to PA in the absence of metal ions. However, the isolated catalytic fragment has a considerably weaker affinity for PA surfaces. In contrast to PA surfaces, all PLD forms exhibited very low affinity for PC interfaces with an increased binding when Ba(2+) was added. All PLD forms also bound tightly to other anionic phospholipid surfaces (e.g. phosphatidylserine, phosphatidylinositol, and phosphatidylmethanol). However, this binding was not modulated in the same way by divalent cations. Chemical cross-linking studies suggested that a major effect of PLD binding to PA.Ca(2+) surfaces is aggregation of the enzyme. These results indicate that PLD partitioning to phospholipid surfaces and kinetic activation are two separate events and suggest that the Ca(2+) modulation of PA.PLD binding involves protein aggregation that may be the critical interaction for activation.

Barium↗

PubMed1999

Effects of osmotic stress on Methanococcus thermolithotrophicus: 13C-edited 1H-NMR studies of osmolyte turnover.

In vivo NMR studies of the thermophilic archaeon Methanococcus thermolithotrophicus, with sodium formate as the substrate for methanogenesis, were used to monitor formate utilization, methane production, and osmolyte pool synthesis and turnover under different conditions. The rate of formate conversion to CO2 and H2 decreased for cells adapted to higher external NaCl, consistent with the slower doubling times for cells adapted to high external NaCl. However, when cells grown at one NaCl concentration were resuspended at a different NaCl, formate utilization rates increased. Production of methane from 13C pools varied little with external NaCl in nonstressed culture, but showed larger changes when cells were osmotically shocked. In the absence of osmotic stress, all three solutes used for osmotic balance in these cells, l-alpha-glutamate, beta-glutamate, and Nepsilon-acetyl-beta-lysine, had 13C turnover rates that increased with external NaCl concentration. Upon hyperosmotic stress, there was a net synthesis of alpha-glutamate (over a 30-min time-scale) with smaller amounts of beta-glutamate and little if any of the zwitterion Nepsilon-acetyl-beta-lysine. This is a marked contrast to adapted growth in high NaCl where Nepsilon-acetyl-beta-lysine is the dominant osmolyte. Hypoosmotic shock selectively enhanced beta-glutamate and Nepsilon-acetyl-beta-lysine turnover. These results are discussed in terms of the osmoadaptation strategies of M. thermolithotrophicus.

Carbon Isotopes↗

PubMed1999

A 20-kDa domain is required for phosphatidic acid-induced allosteric activation of phospholipase D from Streptomyces chromofuscus.

Two phospholipase D (PLD) enzymes with both hydrolase and transferase activities were isolated from Streptomyces chromofuscus. There were substantial differences in the kinetic properties of the two PLD enzymes towards monomeric, micellar, and vesicle substrates. The most striking difference was that the higher molecular weight enzyme (PLD57 approximately 57 kDa) could be activated allosterically with a low mole fraction of phosphatidic acid (PA) incorporated into a PC bilayer (Geng et al., J. Biol. Chem. 273 (1998) 12195-12202). PLD42/20, a tightly associated complex of two peptides, one of 42 kDa and the other 20 kDa, had a 4-6-fold higher Vmax toward PC substrates than PLD57 and was not activated by PA. N-Terminal sequencing of both enzymes indicated that both components of PLD42/20 were cleavage products of PLD57. The larger component included the N-terminal segment of PLD57 and contained the active site. The N-terminus of the smaller peptide corresponded to the C-terminal region of PLD57; this peptide had no PLD activity by itself. Increasing the pH of PLD42/20 to 8.9, followed by chromatography of PLD42/20 on a HiTrap Q column at pH 8.5 separated the 42- and 20-kDa proteins. The 42-kDa complex had about the same specific activity with or without the 20-kDa fragment. The lack of PA activation for the 42-kDa protein and for PLD42/20 indicates that an intact C-terminal region of PLD57 is necessary for activation by PA. Furthermore, the mechanism for transmission of the allosteric signal requires an intact PLD57.

Allosteric Site↗

PubMed1999

Action of phosphatidylinositol-specific phospholipase Cgamma1 on soluble and micellar substrates. Separating effects on catalysis from modulation of the surface.

The kinetics of PI-PLCgamma1 toward a water-soluble substrate (inositol 1,2-cyclic phosphate, cIP) and phosphatidylinositol (PI) in detergent mixed micelles were monitored by 31P NMR spectroscopy. That cIP is also a substrate (Km = approximately 15 mM) implies a two-step mechanism (intramolecular phosphotransferase reaction to form cIP followed by cyclic phosphodiesterase activity to form inositol-1-phosphate (I-1-P)). PI is cleaved by PI-PLCgamma1 to form cIP and I-1-P with the enzyme specific activity and ratio of products (cIP/I-1-P) regulated by assay temperature, pH, Ca2+, and other amphiphilic additives. Cleavage of both cIP and PI by the enzyme is optimal at pH 5. The effect of Ca2+ on PI-PLCgamma1 activity is unique compared with other isozymes enzymes: Ca2+ is necessary for the activity and low Ca2+ activates the enzyme; however, high Ca2+ inhibits PI-PLCgamma1 hydrolysis of phosphoinositides (but not cIP) with the extent of inhibition dependent on pH, substrate identity (cIP or PI), substrate presentation (e.g. detergent matrix), and substrate surface concentration. This inhibition of PI-PLCgamma1 by high Ca2+ is proposed to derive from the divalent metal ion-inducing clustering of the PI and reducing its accessibility to the enzyme. Amphiphilic additives such as phosphatidic acid, fatty acid, and sodium dodecylsulfate enhance PI cleavage in micelles at pH 7.5 but not at pH 5.0; they have no effect on cIP hydrolysis at either pH value. These different kinetic patterns are used to propose a model for regulation of the enzyme. A key hypothesis is that there is a pH-dependent conformational change in the enzyme that controls accessibility of the active site to both water-soluble cIP and interfacially organized PI. The low activity enzyme at pH 7.5 can be activated by PA (or phosphorylation by tyrosine kinase). However, this activation requires lipophilic substrate (PI) present because cIP hydrolysis is not enhanced in the presence of PA.

Animals↗

PubMed1999

Characterization of a tetrameric inositol monophosphatase from the hyperthermophilic bacterium Thermotoga maritima.

Inositol monophosphatase (I-1-Pase) catalyzes the dephosphorylation step in the de novo biosynthetic pathway of inositol and is crucial for all inositol-dependent processes. An extremely heat-stable tetrameric form of I-1-Pase from the hyperthermophilic bacterium Thermotoga maritima was overexpressed in Escherichia coli. In addition to its different quaternary structure (all other known I-1-Pases are dimers), this enzyme displayed a 20-fold higher rate of hydrolysis of D-inositol 1-phosphate than of the L isomer. The homogeneous recombinant T. maritima I-1-Pase (containing 256 amino acids with a subunit molecular mass of 28 kDa) possessed an unusually high V(max) (442 micromol min(-1) mg(-1)) that was much higher than the V(max) of the same enzyme from another hyperthermophile, Methanococcus jannaschii. Although T. maritima is a eubacterium, its I-1-Pase is more similar to archaeal I-1-Pases than to the other known bacterial or mammalian I-1-Pases with respect to substrate specificity, Li(+) inhibition, inhibition by high Mg(2+) concentrations, metal ion activation, heat stability, and activation energy. Possible reasons for the observed kinetic differences are discussed based on an active site sequence alignment of the human and T. maritima I-1-Pases.

Enzyme Stability↗

PubMed1998

Nonessential activation and competitive inhibition of bacterial phosphatidylinositol-specific phospholipase C by short-chain phospholipids and analogues.

Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis is an allosteric enzyme with both a phospholipid activator site and an active site. The activation of PI-PLC enzyme is optimal with phosphatidylcholine (PC) binding to the activator site and anchoring the enzyme to the interface [Zhou, C., et al. (1997) Biochemistry 36, 347-355; Zhou, C., et al. (1997) Biochemistry 36, 10089-10091]. In contrast to PC, anionic short-chain phospholipids with smaller headgroups [phosphatidylmethanol (PMe) and phosphatidic acid (PA)] as well as phosphatidylglycerol (PG) can bind to both sites playing dual roles: nonessential activation and competitive inhibition of cyclic-(1, 2)-inositol phosphate hydrolysis. PG is also a substrate, albeit a poor one, for PI-PLC, and is cleaved slowly to form alpha-glycerol phosphate. Analysis of enzyme kinetics using cIP as the substrate coupled with effects of different short-chain phospholipids on enzyme intrinsic fluorescence indicates that anionic phospholipids with small headgroups bind to the two sites with different affinities. If no interface is present, all dihexanoylphospholipids bind to the activator site more strongly than to the active site. When the activator site is occupied, it is likely that the enzyme undergoes a conformational change that allows phospholipids to bind easily to the active site. Such behavior is consistent with the observation that enzyme activation is detected at low short-chain anionic phospholipid concentrations with inhibition observed at higher concentrations, and that only inhibition is seen with these phospholipids added as monomers in the presence of a PC interface that optimally activates the PI-PLC. A kinetic model is used to extract the affinity of short-chain lipids for the active site from experimental data.

Bacillus thuringiensis↗

PubMed1998

Activation of phospholipase D by phosphatidic acid. Enhanced vesicle binding, phosphatidic acid-Ca2+ interaction, or an allosteric effect?

The activity of bacterial phospholipase D (PLD), a Ca2+-dependent enzyme, toward phosphatidylcholine bilayers was enhanced 7-fold by incorporation of 10 mol % phosphatidic acid (PA) in the vesicle bilayer. Addition of other negatively charged lipids such as phosphatidylinositol, phosphatidylmethanol, and oleic acid either inhibited or had no effect on enzyme activity. Only negatively charged lipids with a free phosphate group, phosphatidylinositol 4-phosphate and lyso-PA, had the same effect as PA on enzyme activity. Changes in vesicle curvature and fusion were not the reason for PA activation; rather, a metal ion-induced lateral segregation of PA in the vesicle bilayer correlated with PLD activation. Significant PA activation was also observed with monomer phosphatidylcholine substrate upon the addition of PA vesicles. The PA activation was caused by Ca2+.PA interacting with PLD at an allosteric site other than active site.

Allosteric Regulation↗

PubMed1998

Phosphatidylcholine activation of bacterial phosphatidylinositol-specific phospholipase C toward PI vesicles.

The effect of different phospholipids on the kinetic behavior of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis toward PI vesicles has been investigated. Cosonicated PC/PI vesicles displayed enhanced hydrolysis of PI when less than 0. 20 mole fraction PC was incorporated into the vesicle; higher mole fractions of PC led to a decrease from the maximum activity mimicking surface dilution of substrate. Since the PC could affect PI-PLC binding to vesicles, the effect of separate PC vesicles on enzymatic hydrolysis of PI vesicles was examined. Separate phosphatidylcholine vesicles were found to activate PI-PLC-catalyzed cleavage of PI vesicles up to 7-fold. The activation was completely abolished when the PC vesicle was composed of cross-linked molecules. In the absence of enzyme, fluorescence resonance energy transfer studies did not detect any fusion between PI and PC vesicles if the total lipid concentration was below 2 mM. Higher total lipid concentrations (>20 mM) increased PC transfer between PC and PI vesicles, producing a PI vesicle population with small amounts of PC in the outer monolayer. This suggested that the activation of PI-PLC toward PI vesicles reflects the time scale of transfer of PC from PC vesicles to PI vesicles. Cosonicated PC/PI vesicles provide a measure of enzyme activity versus mole fraction of PC that can be used to estimate the extent of vesicle exchange or fusion between separate vesicle pools. The effects of other phospholipid vesicles on PI-PLC hydrolysis of PI were also examined; zwitterionic lipids were activators while anionic phospholipids inhibited activity. The results indicated that PC molecules in the PI interface allosterically bind to PI-PLC and help anchor enzyme in a more active conformation to the PI interface.

Bacillus thuringiensis↗

PubMed1998

Engineering of the nonspecific phospholipase C from Bacillus cereus: replacement of glutamic acid-4 by alanine results in loss of interfacial catalysis and enhanced phosphomonoesterase activity.

The nonspecific phospholipase C from Bacillus cereus is a zinc metalloenzyme that catalyzes the hydrolysis of phospholipids to yield diacylglycerol and a phosphate monoester. Glu-4 has been proposed as a potential candidate for the general base in the hydrolysis reaction and was shown to interact with the substrate headgroup. Site-specific mutagenesis studies suggest that Glu-4 is important for substrate binding but not for catalysis. This residue is also critical for the enzyme's preference for a phosphodiester substrate. PA, both monomeric and micellar, is shown to be a poor substrate and inhibitor of wild-type PLC. When Glu-4 was mutated to an alanine, a significant increase in PA hydrolysis and a decrease in PC hydrolysis were observed. Unlike the wild type, kinetic studies suggest that the Glu-4-->Ala mutant does not exhibit interfacial activation and processive catalysis. Glu-4 is part of a highly flexible loop flanking the entrance to the active site, suggesting that this loop might constitute an interfacial binding recognition site. This is the first evidence for the presence of an interfacial binding site distinct from the active site in the nonspecific PLC.

Alanine↗