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A Peterkofsky

Publications and source records attributed to A Peterkofsky.

At least 19 recordsLinked to original sources
PubMed1992

Requirement for GLY-60 of Escherichia coli adenylyl cyclase for ATP binding and catalytic activity.

The region of Escherichia coli adenylyl cyclase spanned by glycine-55 to threonine-65 was tested for its importance for enzyme activity. Site-directed mutagenesis was used to replace glycine-55 and glycine-60 as well as lysine-59, leucine-63 and threonine-65 with other amino acids. While substitution of glycine-55 with aspartic acid produced no significant change in kinetic parameters, the change of glycine-60 to aspartic acid or asparagine eliminated binding to 8-azido-ATP and decreased the Vmax (two orders of magnitude) and Km (factor of four-five). Smaller effects on kinetic parameters were observed with substitutions of lysine-59, leucine-63 or threonine-65.

Adenosine Triphosphate↗
PubMed1991

Characterization and generation of Escherichia coli adenylate cyclase deletion mutants.

Escherichia coli delta cya-283 is a 75-bp in-frame deletion overlapping the 5' end of delta cya-854; delta cya-201 is a 41-bp frameshift deletion overlapping the 3' end of delta cya-854. Sequence repeats were found at the boundaries of delta cya-283 and delta cya-201, suggesting a mechanism for deletion formation. Recombinant DNA procedures were used to construct a strain in which the total cya structural gene in the chromosome was replaced by the kanamycin resistance gene.

Adenylyl Cyclases↗
PubMed1989

Hyperexpression and purification of Escherichia coli adenylate cyclase using a vector designed for expression of lethal gene products.

We describe the construction of a new generation of vectors (pRE) for the hyperexpression of lethal gene products such as adenylate cyclase in Escherichia coli. The pRE vectors are based on the lambda PL promoter and lambda cII ribosome binding site described by Shimatake and Rosenberg (Nature, 292, 128-132, 1981). They have a unique NdeI restriction endonuclease site 3' of the lambda cII ribosome binding site that includes the ATG initiation codon, multilinker cloning sites 3' to the NdeI site, and two lambda transcription terminators 5' and 3' of the lambda PL promoter to eliminate nonspecific transcription and reduce leaky PL transcription, respectively. For hyperexpression of adenylate cyclase, tight control of transcription was necessary since elevation of cAMP levels above the physiological range is lethal to E. coli. Lethality associated with the overproduction of adenylate cyclase was shown to be mediated through the cAMP receptor protein. We used this expression system to overproduce adenylate cyclase 7500 fold, corresponding to 30% of the total cellular protein. Under these conditions the enzyme precipitated with significant loss of activity. Reducing the rate and amount of adenylate cyclase expression to 16% of the total cell protein produced one fourth of the enzyme in a soluble form with high specific activity. The soluble adenylate cyclase was purified to near homogeneity.

Adenylyl Cyclases↗
PubMed1989

Crystal structure of a cAMP-independent form of catabolite gene activator protein with adenosine substituted in one of two cAMP-binding sites.

Catabolite gene activator protein (CAP) in the presence of cAMP stimulates transcription from several operons in Escherichia coli. A cAMP-independent variant, in which Ala-144 is replaced by Thr (CAP91), is activated by analogues of cAMP, such as adenosine, which do not activate the wild-type CAP. In order to test the effect of adenosine on the structure, a crystal of CAP91 grown as a complex with cAMP was soaked in a solution of 10 mM adenosine, and X-ray diffraction data were measured to 3.5-A resolution. The difference Fourier map calculated with phases from the CAP91 structure showed significant negative density at the position of the phosphate of cAMP bound in one subunit of the CAP91 dimer. Adenosine was preferentially substituted for cAMP in the subunit in the "closed" conformation, while the cAMP-binding site of the "open" subunit was apparently still occupied by cAMP. The structure was refined by restrained least-squares methods to an R factor of 20.2%. Adenosine is not bound in exactly the same position as cAMP; instead, the 5'-OH of adenosine is in a new position that allows formation of two hydrogen bonds with Ser-83, replacing two of the three interactions of the phosphate of cAMP with Arg-82 and Ser-83.

Adenosine↗
PubMed1989

Mechanistic and physiological consequences of HPr(ser) phosphorylation on the activities of the phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: studies with site-specific mutants of HPr.

The bacterial phosphotransferase system (PTS) catalyzes the transport and phosphorylation of its sugar substrates. The protein-kinase-catalyzed phosphorylation of serine 46 in the phosphocarrier protein, HPr, inhibits PTS activity, but neither the mechanism of this inhibition nor its physiological significance is known. Site-specific HPr mutants were constructed in which serine 46 was replaced by alanine (S46A), threonine (S46T), tyrosine (S46Y) or aspartate (S46D). The purified S46D protein exhibited markedly lower Vmax and higher Km values than the wild-type, S46T or S46A protein for the phosphoryl transfer reactions involving HPr(His approximately P). Interactions of HPr with the enzymes catalyzing phosphoryl transfer to and from HPr regulated the kinase-catalyzed reaction. These results establish the inhibitory effect of a negative charge at position 46 on PTS-mediated phosphoryl transfer and suggest that HPr is phosphorylated on both histidyl and seryl residues by enzymes that recognize its tertiary rather than its primary structure. In vivo studies showed that a negative charge on residue 46 of HPr strongly inhibits PTS-mediated sugar uptake, but that competition of two PTS permeases for HPr(His approximately P) is quantitatively more important to the regulation of PTS function than serine 46 phosphorylation.

Amino Acids↗
PubMed1989

Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

The phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli constitutes a major pathway for sugar translocation. It is composed of integral membrane proteins (enzyme II components) that recognize specific extracellular sugars as well as phosphocarrier proteins, one of which is called enzyme I. While enzyme I plays a role in energizing the enzyme II for sugar transfer, its precise cellular distribution had not previously been defined. This study was designed to elucidate the cellular location of this protein by immunoelectron microscopy. Enzyme I antibody bound to E. coli cryosections was visualized with protein A-gold. The gold particles in sections of wild-type E. coli were found primarily associated with the surface of the inner membrane. A strain of E. coli harboring a plasmid encoding the gene for enzyme I was also tested for its distribution of enzyme I. Consistent with the biochemically established overproduction of enzyme I, this strain showed an approximately 80-fold higher density of gold particles per unit cell volume than the wild-type cells. The substantial overproduction of immunoreactive enzyme I was associated with a significant (approximately 20-fold) increase in the amount of that protein bound to the inner membrane. In addition, a substantial fraction of the total enzyme I accumulated within a 60-nm-wide zone in the vicinity of the inner membrane. A model to explain the zonal distribution of enzyme I under conditions of overexpression of the protein is presented.

Cell Membrane↗
PubMed1988

Protein phosphorylation in Mycoplasma gallisepticum.

Incubation of the soluble fraction derived from Mycoplasma gallisepticum cells with [gamma-32P]ATP results in the phosphorylation of several endogenous proteins. One protein with an apparent molecular mass of 55 kDa was the acceptor of more than 95% of the radioactive phosphate. This protein was also found to be radiolabeled in intact cells grown in the presence of [32P]orthophosphate. Acid hydrolysis of the phosphorylated 55-kDa protein followed by two-dimensional electrophoresis revealed that the 32P-labeled material co-migrated with phosphoserine. The in vitro phosphorylation of the 55-kDa protein has an optimum pH of 5.5-6.0 and is not affected by various metabolites of glycolysis, by cAMP or by calmodulin with or without Ca2+. The phosphorylation is dependent upon divalent cations, a dependency that is best fulfilled by the simultaneous addition of Ca2+ and Zn2+ that act in a specific and cooperative manner. Of a variety of possible exogenous protein acceptors tested, the endogenous protein kinase was capable to phosphorylate only phosvitin. The phosphorylation of the 55-kDa protein is reversible through the activity of a phosphoprotein phosphatase present in the soluble fraction of M. gallisepticum. The phosphoprotein phosphatase has an optimum pH of 7.5-8.0, is inhibited by NaF and stimulated to a large extent by inorganic phosphate and arsenate and to a lesser extent by pyrophosphate ATP and ADP. The possible association of the reversible protein phosphorylation to cell shape and gliding motility of M. gallisepticum are discussed.

Adenosine Triphosphate↗
PubMed1988

Arginine substituted for leucine at position 195 produces a cyclic AMP-independent form of the Escherichia coli cyclic AMP receptor protein.

Mutant forms (CRP*) of the Escherichia coli cAMP receptor protein (CRP) that activate CRP-dependent promoters in the absence of the normal allosteric effector (cAMP) have been described. A previous report (Harman, J. G., McKenney, K., and Peterkofsky, A. (1986) J. Biol. Chem. 261, 16332-16339) detailed the properties of three CRP* mutant proteins. One protein, 220 CRP, has amino acid substitutions at positions 127 and 170 and low CRP* activity in vivo. A second protein, 222 CRP, has the amino acid substitutions present in 220 CRP and a third substitution (arginine for leucine) at position 195. 222 CRP has high CRP* activity in vivo and high apparent affinity for lacP DNA relative to the 220 CRP in vitro. In this report, we evaluate the effect of a single amino acid substitution at position 195 (leucine to arginine) on CRP activity both in vivo and in vitro. Cells (cya delta crp delta/pJH8crpR195) containing R195 CRP were found to exhibit a CRP* phenotype, expressing a variety of CRP-dependent genes in the absence of added cAMP. R195 CRP exhibited both CRP* activity in vitro and increased apparent affinity for cAMP relative to wild-type CRP. CRP titration experiments performed using an in vitro lac transcription system suggest that the isolated substitution of arginine at position 195 does not confer on CRP the high lacP affinity that distinguishes the 220 and 222 forms of CRP. These findings lead us to the conclusion that the effects of multiple mutations in CRP can be both cumulative and interactive.

Arginine↗
PubMed1988

Redistribution of phosphate pools and the regulation of Escherichia coli adenylate cyclase activity.

The enzyme adenylate cyclase plays a key role in mediating the phenomenon of catabolite repression in Escherichia coli. The mechanism by which one sugar prevents the expression of the gene for another catabolite depends on the capacity of the cell to take up the sugar. Sugars that are most effective in the repression mechanism are those that are transported by the phosphoenolpyruvate-energized phosphotransferase system. The hypothesis presented here is that one or more of the proteins associated with this sugar transport system interact with adenylate cyclase and, when they are in their phosphorylated form, activate the enzyme, provided other factors that permit this activation are present. Another essential activator of adenylate cyclase is inorganic orthophosphate. When E. coli are starved for sugars, the pool of total phosphate is accounted for primarily as inorganic orthophosphate, ATP, phosphoenolpyruvate, and transport proteins in their phospho-forms, a condition that promotes activation of adenylate cyclase. When cells are exposed to sugars, the phosphate pool becomes drastically redistributed, such that the level of inorganic orthophosphate and transport phosphoproteins decreases markedly while the pool of sugar phosphate increases. This translation of the extracellular availability of carbon sources into an intracellular phosphate redistribution is the immediate event that is responsible for catabolite repression.

Adenylyl Cyclases↗
PubMed1988

Evidence for the presence of heat-stable protein (HPr) and ATP-dependent HPr kinase in heterofermentative lactobacilli lacking phosphoenolpyruvate:glycose phosphotransferase activity.

An analysis of the biochemical basis for the lack of phosphoenolpyruvate:glycose phosphotransferase activity in heterofermentative lactobacilli was carried out. Extracts of Lactobacillus brevis and Lactobacillus buchneri failed to reconstitute phosphotransferase activity of extracts of Staphylococcus aureus mutants impaired in the phosphotransferase system due to the absence of enzyme I, enzyme IILac, or enzyme IIILac activity, suggesting that these lactobacilli lack those phosphotransferase system components. In contrast, complementation tests with an extract of a S. aureus mutant deficient in heat-stable protein (HPr) indicated the presence of HPr activity in heterofermentative lactobacilli. The HPr of L. brevis was purified and shown to have properties similar to those of a typical HPr. In addition, L. brevis possesses an ATP-dependent protein kinase that phosphorylates a serine residue of the endogenous HPr as well as other HPrs of Gram-positive origin. The kinase activity is markedly stimulated by phosphorylated compounds related to sugar metabolism and is negatively modulated by orthophosphate, pyrophosphate, or arsenate and by a low molecular weight endogenous factor. In keeping with the idea of a regulatory role for the phosphorylation of HPr in lactobacilli, a HPr[Ser(P)] phosphatase activity in L. brevis was also demonstrated. On the basis of the finding of HPr and a system for its reversible covalent modification in an organism devoid of a functional phosphotransferase system we propose that, in lactobacilli, HPr has a role in the regulation of pathways other than the phosphotransferase system.

Arsenates↗
PubMed1987

Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein.

Escherichia coli NCR91 synthesizes a mutant form of catabolite gene activator protein (CAP) in which alanine 144 is replaced by threonine. This mutant, which also lacks adenylate cyclase activity, has a CAP phenotype; in the absence of cAMP it is able to express genes that normally require cAMP. CAP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild type CAP X cAMP complex. X-ray diffraction data were measured to 2.4-A resolution and the CAP91 structure was determined using initial model phases from the wild type structure. A difference Fourier map calculated between CAP91 and wild type showed the 2 alanine to threonine sequence changes in the dimer and also a change in orientation of cysteine 178 in one of the subunits. The CAP91 coordinates were refined by restrained least squares to an R factor of 0.186. Differences in the atomic positions of the wild type and mutant protein structures were analyzed by a vector averaging technique. There were small changes that included concerted motions in the small domains, in the hinge between the two domains and in an adjacent loop between beta-strands 4 and 5. The mutation at residue 144 apparently causes changes in the position of some protein atoms that are distal to the mutation site.

Cyclic AMP↗
PubMed1987

Regulation of beta-galactoside transport and accumulation in heterofermentative lactic acid bacteria.

Galactose-grown cells of the heterofermentative lactic acid bacteria Lactobacillus brevis and Lactobacillus buchneri transported methyl-beta-D-thiogalactopyranoside (TMG) by an active transport mechanism and accumulated intracellular free TMG when provided with an exogenous source of energy, such as arginine. The intracellular concentration of TMG resultant under these conditions was approximately 20-fold higher than that in the medium. In contrast, the provision of energy by metabolism of glucose, gluconate, or glucosamine promoted a rapid but transient uptake of TMG followed by efflux that established a low cellular concentration of the galactoside, i.e., only two- to fourfold higher than that in the medium. Furthermore, the addition of glucose to cells preloaded with TMG in the presence of arginine elicited a rapid efflux of the intracellular galactoside. The extent of cellular TMG displacement and the duration of the transient effect of glucose on TMG transport were related to the initial concentration of glucose in the medium. Exhaustion of glucose from the medium restored uptake and accumulation of TMG, providing arginine was available for ATP generation. The nonmetabolizable sugar 2-deoxyglucose elicited efflux of TMG from preloaded cells of L. buchneri but not from those of L. brevis. Phosphorylation of this glucose analog was catalyzed by cell extracts of L. buchneri but not by those of L. brevis. Iodoacetate, at a concentration that inhibits growth and ATP production from glucose, did not prevent efflux of cellular TMG elicited by glucose. The results suggested that a phosphorylated metabolite(s) at or above the level of glyceraldehyde-3-phosphate was required to evoke displacement of intracellular TMG from the cells. Counterflow experiments suggested that glucose converted the active uptake of TMG in L. brevis to a facilitated diffusion mechanism that allowed equilibrium of TMG between the extra- and intracellular milieux. The means by which glucose metabolites elicited this vectorial regulation is not known, but similarities to the inducer expulsion that has been described for homofermentative Streptococcus and Lactobacillus species suggested the involvement of HPr, a protein that functions as a phosphocarrier protein in the phosphotransferase system, as well as a presumptive regulator of sugar transport. Indeed, complementation assays wit extracts of Staphylococcus aureus ptsH mutant revealed the presence of HPr in L. brevis, although this lactobacillus lacked a functional phaosphoenolpyruvate-dependent phosphortransferase system for glucose, 2-deoxyglucose, or TMG.

Arginine↗
PubMed1986

Inhibition of E. coli adenylate cyclase activity by inorganic orthophosphate is dependent on IIIglc of the phosphoenolpyruvate:glycose phosphotransferase system.

The relationship of adenylate cyclase, inorganic orthophosphate and the proteins of the phosphoenolpyruvate:glycose phosphotransferase system (PTS) was studied. A strain deleted for the genes for Enzyme I and IIIglc of the PTS was transformed with plasmids expressing either Enzyme I and HPr, IIIglc or all three proteins. The fully reconstituted strain showed a Pi-dependent stimulation of adenylate cyclase activity; in contrast, the strain expressing only IIIglc showed a Pi-dependent inhibition of adenylate cyclase activity.

Adenylyl Cyclase Inhibitors↗
PubMed1986

Structure-function analysis of three cAMP-independent forms of the cAMP receptor protein.

cAMP receptor protein (CRP)-dependent operon expression in Escherichia coli requires the CRP X cAMP complex form of wild-type CRP. One class of crp mutants (crp*) activates CRP-dependent promoters in strains (cya) incapable of endogenous cAMP synthesis. Of fundamental interest is the difference in regulatory properties exhibited by crp* mutant strains, some of which exhibit glucose-mediated repression of beta-galactosidase synthesis, some of which do not. To gain a better understanding of the mechanisms of cAMP-independent promoter activation and repression we have: determined through cloning and DNA sequence analysis the primary structure of three CRP* forms of CRP; purified the mutant proteins; characterized the effect of these mutations on CRP secondary structure; and studied CRP*-activated lac promoter regulation in a purified in vitro transcription system. The results of this study provide strong evidence that mutations in crp alter the conformation of CRP and result in cAMP-independent activation of CRP-dependent promoters in vitro. In addition, a CRP allele-specific inhibition of CRP* activity by spermidine was observed in vitro that parallels crp* strain-specific sensitivity to glucose-mediated repression of CRP-dependent enzyme synthesis in vivo. This observation provides evidence that catabolite repression in cells lacking cAMP may be mediated through a mechanism that inhibits CRP* activity.

Bacterial Proteins↗
PubMed1986

Stimulation of Escherichia coli adenylate cyclase activity by elongation factor Tu, a GTP-binding protein essential for protein synthesis.

A unique feature of eucaryotic adenylate cyclases is their interaction with GTP-binding proteins that mediate hormonal responses. Until now, there has been no evidence for regulation of Escherichia coli adenylate cyclase by a GTP-binding protein. We describe here that the most abundant protein in E. coli, the GTP-binding protein EF-Tu, which is important as an elongation factor in protein synthesis, also serves as a stimulator of adenylate cyclase activity. Homogeneous EF-Tu specifically increased the activity of purified adenylate cyclase as much as 70%; other E. coli GTP-binding proteins had no effect on enzyme activity. A study of the guanine nucleotide specificity for EF-Tu-mediated stimulation of adenylate cyclase activity suggested that the preferred activator is EF-Tu X GDP. To account for the GTP-specific stimulation of adenylate cyclase activity observed in intact cells, we propose that the nucleotide specificity for EF-Tu-dependent activation of adenylate cyclase is governed by other factors in the cell.

Adenylyl Cyclases↗
PubMed1985

The Escherichia coli adenylate cyclase complex. Stimulation by potassium and phosphate.

In Escherichia coli, adenylate cyclase activity in toluene-treated cells can be inhibited by glucose while the activity in a broken cell preparation cannot. Adenylate cyclase activity in the permeabilized but not in broken cells is stimulated somewhat specifically and additively by potassium and phosphate. Kinetic studies show sigmoid substrate-velocity curves for the toluene-treated cells but hyperbolic curves for the broken cells. The stimulatory effects of potassium and phosphate on adenylate cyclase activity in tolulene-treated cells are associated with increases in the Vmax and Km for ATP. While the enzyme activity in toluene-treated cells shows a preference for magnesium over manganese, the reverse is observed in broken cells. Stimulation of adenylate cyclase activity in toluene-treated cells requires the presence of the proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The PTS proteins can be phosphorylated in a P-enolpyruvate-dependent reaction. The stimulatory effects of ions will not occur if the PTS proteins are not phosphorylated. Since potassium phosphate stimulates both adenylate cyclase and PTS activities in toluene-treated cells, it is proposed that the effect of potassium phosphate on adenylate cyclase activity is mediated through an effect on the PTS. A model for dual regulation by glucose of adenylate cyclase activity is proposed. This model involves regulation of both the condition of the PTS proteins as well as the cellular concentration of phosphate.

Adenylyl Cyclases↗
PubMed1985

Translational efficiency of the Escherichia coli adenylate cyclase gene: mutating the UUG initiation codon to GUG or AUG results in increased gene expression.

Roy et al. [Roy, A., Haziza, C. & Danchin, A. (1983) EMBO J. 2, 791-797] established that translation of Escherichia coli adenylate cyclase initiates at a UUG codon, and they suggested this might decrease the efficiency of translation. We investigated the effect of varying the initiation codon on the expression of the adenylate cyclase (cya) gene. Using oligonucleotide-directed mutagenesis, we changed the UUG initiation codon to GUG and the more common initiator AUG and assayed for cya gene expression in a number of ways. First, the GUG initiation codon, in place of UUG, doubled cya expression when cya was expressed from the dual cya P1/P2 promoters. The corresponding AUG codon construct was nonviable. Second, when the cya gene was placed under the transcriptional control of the thermoinducible phage lambda PL promoter, the relative amounts of cya gene product were 1:2:6 for the UUG, GUG, and AUG initiation codons, respectively. Finally, the cya P2 promoter, Shine-Dalgarno sequence, and the DNA corresponding to the first 86 codons of cya were fused to DNA encoding the E. coli galactokinase gene beginning at the second codon. The relative amounts of the fusion polypeptides, which had galactokinase activity, were 1:2:3 for the UUG, GUG, and AUG initiation codons, respectively. These results demonstrate that the cya UUG initiation codon limits cya expression at the level of translation.

Adenylyl Cyclases↗