David P. Bloxham

Muscle and liver pyruvate kinases are closely related: amino acid sequence comparisons

Previous evidence has shown that the M1 and L pyruvate kinase isozymes differ markedly in kinetic and immunological properties, amino acid compositions and peptide maps. However, the amino acid sequence results we present here for the N‐terminal region and for a region of the C domain show that the M1 and L isozymes are very similar. The variable length of the N‐terminal sequences also explains the difference in regulation by phosphorylation between the M1 and L isozymes. The M1 isozyme lacks the serine residue that has been shown to be phosphorylated in the L isozyme.

An anti-anabolic role of adenosine 3',5'-cyclic monophosphate in the control of liver metabolism. a hypothetical mechanism for gluconeogenesis☆

Abstract 1. 1. It is shown that in rat liver slices cyclic AMP caused a co-ordinated inhibition of protein, fatty acid, and cholesterol synthesis (anti-anabolic) from several radioactive precursors. 2. 2. The inhibition of cholesterol and fatty acid synthesis was due to a genuine decrease in the activity of the biosynthetic pathway. 3. 3. Under conditions when the anti-anabolic effect was obtained, the incorporation of [14CJbicarbonate into glucose was increased, confirming that cyclic AMP favours glyconeogencsis. 4. 4. The mechanism by which cyclic AMP produced these effects was studied further by the use of cell-free preparations from rat liver. Cyclic AMP was shown to inhibit protein synthesis in a postmitochondrial supernatant. This inhibition only occurred in the presence of an excess of the postmicrosomal supernatant fraction. These results are discussed on the basis that cyclic AMP might interact with a component in the posticrosomal fraction capable of modifying the activity of the protein synthetic machinery. 5. 5. Examples are provided to suggest that this anti-anabolic effect of cyclic AMP may operate in vivo.

Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate

Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.

Identification of subsidiary catalytic groups at the active site of β-ketoacyl-CoA thiolase by covalent modification of the protein

beta-Ketoacyl-CoA thiolase (acyl-CoA:acetyl-CoA C-acyltransferase, EC 2.3.1.16) is known to possess sulfhydryl groups of cysteines at the active site that are essential for its catalytic activity. Other groups at the active site that participate in the catalytic process were identified by using anhydride reagents which covalently modify the protein by specifically reacting with any amino groups potentially present at the active site. Since these reagents may also react with thiol groups, the enzymes amino groups were modified after masking the cysteine thiols present by an alkylalkane thiosulfonate-type reagent, methyl methanethiol-sulfonate (MMTS), that selectively formed a disulfide bridge, thus generating an inactive thiolmethylated enzyme. When this procedure was followed, the enzyme could be undoubtedly modified at its amino by the anhydride reagent, leading to a doubly modified protein. The thiomethyl group could then be removed by reduction with dithiothreitol, yielding an enzyme modified solely on the amino residues. The amino group could be unblocked in turn by exposure to acidic pH. The different anhydrides inactivated thiolase, but only acetoacetyl coenzyme A (AcAcCoA) provided any protection against inactivation. When thiolmethylcitraconyl thiolase was reduced with dithiothreitol the enzyme remained inactive, but when the doubly modified enzyme was exposed to pH 5 then the reduction led to formation of an active enzyme. These results are interpreted as demonstrating a role for an amino group at the enzyme active site. A catalytic mechanism is proposed for the enzyme which involves the amino group.

Citrate synthase activity in Escherichia coli harbouring hybrid plasmids containing the gltA gene.

A hybrid plasmid, pDB2, was constructed by ligating a 3.24 kb EcoRI/HindIII fragment of the Escherichia coli chromosome into pBR322. This was used to transform a gltA mutant which was devoid of citrate synthase activity. The resultant strain expressed very high citrate synthase activity and this enabled a simplified purification of the homogeneous enzyme in high yield. The subunit Mr was estimated as 47000-49000 by SDS gel electrophoresis, which closely resembles the eukaryotic form of the enzyme. Evidence for some conservation of sequence between the two proteins was revealed in the acid cleavage pattern at aspartyl-prolyl residues. In addition to coding for the structural gene for citrate synthase, the 3.24 kb EcoRI/HindIII fragment also retained the genetic structure necessary for control of enzyme synthesis since the expression of enzyme activity in the strain harbouring pDB2 was still subject to glucose repression.

Use of methanethiolation to investigate the catalytic role of sulphydryl groups in rabbit skeletal muscle pyruvate kinase

Incubation of rabbit skeletal muscle pyruvate kinase (ATP:pyruvate 2-O-phosphotransferase, EC 2.7.1.40) with methyl methanethiosulphonate resulted in the time- and inhibitor concentration-dependent loss of enzyme activity. Substrates or products of the catalytic reaction prevented the loss of activity caused by methanethiolation. Their effectiveness as protecting agents was placed in the order ADP greater than ATP greater than Mg2+ greater than phosphoenolpyruvate greater than pyruvate. The essential catalytic cation, K+, had no effect on the methanethiolation reaction. [Me-3H]Methanethiosulphonate modified all the available cysteine thiol groups which correlated to the incorporation of four SC3H3 groups per protomer. Four radioactive peptides were obtained on tryptic peptide mapping. When methanethiolation was carried out in the presence of Mg2+ alone or with Mg2+ and ATP together, then only three SC3H3 groups were incorporated into each subunit. If MgATP protected methanethiolated pyruvate kinase was reacted with iodo[2-3H]acetic acid then 1.37 +/- 0.2 groups per protomer were carboxymethylated. 70% of the radioactivity was located in a single peptide on tryptic peptide mapping. This peptide was isolated and contained the segment carboxymethyl cysteine (Glx, Asx, Ser) Arg. Collectively these data indicate that although all thiol groups are equally accessible to methyl methanethiosulphonate, only a single thiol group participates in the catalytic event. An additional role in the maintenance of structure for this thiol group was also shown in studied of reduction and thermal denaturation of the enzyme.

The use of bacteriophage M13 carrying defined fragments of the Escherichia coli gltA gene to determine the location and structure of the citrate synthase promoter region

SummaryThe gltA gene from Escherichia coli, which encodes citrate synthase, has been located on a 3.24 Kb HindIII/EcoRl restriction fragment. This region contains one restriction site for BamHl and two for BglII. Defined restriction fragments from this region were cloned into suitably cleaved replicative form M13mp8 and M13mp9. The recombinants (M13gltA1 → 10) were isolated as single stranded DNA and characterised on the basis of molecular weight and DNA sequence. The single stranded DNA was converted to the double stranded replicative form and used to transform E. coli strain JM103 from which bacteriophage were isolated. Infection of JM103 with different bacteriophage followed by measurement of expressed citrate synthase activity showed that the complete gltA gene must span the BamHl restriction site, that the control region was on the 5′-terminal side of this restriction site and that the coding region for citrate synthase protein commenced on the 3′-terminal side. Analysis of the DNA sequence of this region allowed us to confirm this model, to identify the start sequence for translation of the structural gene and a number of sequences controlling the initiation of transcription. Of special interest is the fact that there must be an extensive leader sequence (305 nucleotides) separating the predicted sites for initiation of transcription and translation.

Gas chromatographic and gas chromatographic—mass spectrometric characterisation of some thiosulphonates and polymethylene dimethane thiosulphonates

Abstract The characterisation by gas chromatography (GC) and GC—mass spectrometry (MS) of thiosulphonates of the types R1·SO2S·R2 (type I) and CH3·SO2S·(CH2)n S·SO2CH3 (type II) is described. Type I thiosulphonates showed suitable GC—MS properties as the intact molecules. Type II could be characterised by GC—MS only after reduction to the dithiol; direct probe spectra were necessary to characterise all the intact type II compounds by MS.

The requirement for a membrane component to demonstrate the inhibition of cell-free protein synthesis by cyclic AMP

Work from this laboratory has shown that under conditions of enhanced gluconeogenesis, cyclic AMP promotes a coordinated inhibition of hepatic anabolic pathways such as protein, cholesterol and fatty acid biosynthesis [ 1,2] . The mechanisms through which these effects of cyclic AMP are mediated has stimulated much recent interest [3-71. Studies with rat liver slices have shown that cyclic AMP reduces protein synthesis by inhibiting peptide bond formation from ribosome bound aminoacyl tRNA [2]. Similar results were also obtained with relatively crude cell-free systems from liver [2,8,9] . Inhibition, which was dependent upon the presence of ATP and cytosolic protein kinase, resulted in a stable modification of a component of the microsomal fraction PI. The demonstration that purified ribosomal subunits could be phosphorylated in a cyclic AMP and protein kinase dependent reaction [lo] , appeared to provide a potential link between the control of protein synthesis and a cyclic AMP promoted modification of the synthetic apparatus. However, an exhaustive search failed to reveal any change in the synthetic capacity of ribosomes reconstituted from phosphorylated subunits [ 1 l] . On examination of the protein synthetic systems which responded to cyclic AMP, it soon became clear that inhibition was obtained only with cell-free preparations containing a membrane fraction (i.e. microsomes). This prompted us to investigate whether the presence of a membrane component is essential in the response to cyclic AMP. The studies presented in this communication indicate that this is the case.

[47] Chloromethyl ketone derivatives of fatty acids

Publisher Summary This chapter discusses various aspects of chloromethyl ketone derivatives of fatty acids. α-Haloketones are very susceptible to nucleophilic attack, which makes this functional group ideally suited for the design of specific enzyme active-site-directed inhibitors. Classic examples of this use are found in the synthesis of α- N -tosylphenylethylchloromethyl ketone and α-N-tosyllysylchloromethyl ketone as specific inhibitors for proteolytic enzymes. The class I chloromethylketone analogs represent one of the simplest type of analogs of fatty acids containing the reactive halomethyl-ketone functional group. They suffer from the drawback that, chemical manipulation has removed the carboxyl group and these results in a profound solubility change in that class I analogs are virtually insoluble in water. Radioactive inhibitors for binding studies can be conveniently synthesized by acid-catalyzed proton exchange from the methylene groups adjacent to the carbonyl. Potential applications for the chloromethyl ketone fatty acid analogs may be found in all areas of enzymology, involving the binding and metabolism of fatty acids.