Involvement and identification of a lysine in the PPi-site of pyrophosphate-dependent phosphofructokinase from Giardia lamblia.

The substrate binding and/or catalytic site of the pyrophosphate-dependent phosphofructokinase (PPi-PFK) of Giardia lamblia was investigated using an ATP affinity label, 2',3'-dialdehyde of ATP, oxidized ATP (oATP), for the involvement of lysine residues. The enzyme, which uses PPi rather than ATP as a substrate was inhibited by low concentrations of oATP. Oxidized ATP behaves as an affinity label for the substrate binding site as evidenced by saturation kinetics with the formation of reversible complex prior to inactivation, and the observation that the inactivation was stoichiometric with the amount of oATP incorporated which extrapolated to 1 mol per mol of monomeric PPi-PFK. The critical lysine modified by oATP is proposed to be located at the PPi-binding site since complete protection is afforded by PPi; and under steady-state, PPi was competitive with the inhibitor. Other substrates of the reaction in either the forward or reverse direction did not completely protect against inactivation. This is further confirmed by the non-competitive inhibition displayed by either Pi or fructose 1,6, bisphosphate. Furthermore, the Km values for Pi and fructose 1,6 bisphosphate of the oATP-modified enzyme were not altered. The oATP-modified peptides were analyzed by HPLC peptide mapping, and the profile showed a major peak absorbing at 258 nm, which was absent when the modification was carried out in the presence of MgPPi. This peptide was sequenced and found to contain Lys-497. These results suggest that the essential lysine-497 modified by oATP is involved in the binding and/or catalysis of PPi and that an ATP-type of binding domain, with reference to the phosphoryl groups, is present in the PPi-dependent phosphofructokinase of Giardia.

Kinetic mechanisms of polyphosphate glucokinase from Mycobacterium tuberculosis.

Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using inorganic polyphosphates [poly(P)] or ATP. The steady-state kinetic mechanisms of the poly(P)- and ATP-dependent glucokinase reactions were investigated using initial velocity, product inhibition, and dead-end inhibition analyses. In the poly(P)-dependent reaction, the enzyme follows an Ordered Bi Bi sequential mechanism with poly(P) binding to the enzyme first and glucose 6-phosphate dissociating last. Polyphosphate is utilized nonprocessively with a preference for longer chains due to higher kcat/K(m) values. The lack of inhibition at high poly(P) concentrations suggests that binding of poly(P) as a product is not favorable. In the ATP-dependent glucokinase reaction, the data are also consistent with an Ordered Bi Bi sequential mechanism, with ATP binding to the enzyme first and glucose 6-phosphate leaving last. At high concentrations, ATP displays competitive substrate inhibition with respect to glucose, which is consistent with the formation of an enzyme.ATP.ATP nonproductive complex. The overall catalytic efficiencies (kcat/KiaK(b)) of the poly(P)- and ATP-dependent reactions are approximately 10(11) M-2 s-1 and approximately 10(8) M-2 s-1, respectively. The higher catalytic efficiency, high value of the substrate specificity constant (kcat/K(a)) approaching a diffusion-controlled limit, and the absence of substrate inhibition in the poly(P)-dependent reaction suggest that poly(P), rather than ATP, is the major phosphate donor for poly(P)-glucokinase in M. tuberculosis.

Initial rate and equilibrium isotope exchange studies on the ATP-dependent activity of polyphosphate Glucokinase from Propionibacterium shermanii.

Polyphosphate glucokinase [EC 2.7.1.63] catalyzes the phosphorylation of glucose using either inorganic polyphosphate [poly(P)] or ATP as the phosphoryl donor. Both activities purified from Propionibacterium shermanii are the functional properties of a single enzyme with separate binding sites for the two phosphoryl donor substrates. The enzyme was found to utilize poly(P) much more efficiently than it does ATP, with a kcat/Kpoly(P) to kcat/KATP ratio of 2800. The catalytic constant for poly(P) is about 2-fold higher than for ATP. Other nucleotides like GTP and dATP also served as substrates with good efficiencies. The ATP-dependent reaction was analyzed using steady-state kinetics and isotopic exchange kinetics at chemical equilibrium. Intersecting initial velocity patterns for both glucose and ATP indicate sequential addition of substrates. Product inhibition studies resulted in two competitive and two noncompetitive patterns, which is characteristic of a Theorell-Chance mechanism or a random mechanism with two dead-end complexes. Results of isotope exchange experiments, however, rule out a Theorell-Chance mechanism, as well as a truly random mechanism. They are not consistent with a partially random mechanism (although a kinetically compulsory order of substrate binding is not excluded), where glucose is preferentially bound to free enzyme before ATP, and ADP is preferentially released as the first product, followed by glucose 6-phosphate. Dead-end inhibition analysis confirms this order of substrate binding. Competitive inhibition of ADP vs ATP is explained as resulting primarily from binding as a dead-end inhibitor (E.Glc.ADP) and not as a product. Another weaker abortive complex, E.ATP.G6P, is also formed. The chemical transformation or the release of ADP is the rate-limiting step in ATP utilization.

Cloning, expression, and characterization of polyphosphate glucokinase from Mycobacterium tuberculosis.

Polyphosphate glucokinase from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using polyphosphate or ATP as the phosphoryl donor. The M. tuberculosis H37Rv gene encoding this enzyme has been cloned, sequenced, and expressed in Escherichia coli. The gene contains an open reading frame for 265 amino acids with a calculated mass of 27,400 daltons. The recombinant polyphosphate glucokinase was purified 189-fold to homogeneity and shown to contain dual enzymatic activities, similar to the native enzyme from H37Ra strain. The high G+C content in the codon usage (64.5%) of the gene and the absence of an E. coli-like promoter consensus sequence are consistent with other mycobacterial genes. Two phosphate binding domains conserved in the eukaryotic hexokinase family were identified in the polyphosphate glucokinase sequence, however, "adenosine" and "glucose" binding motifs were not apparent. In addition, a putative polyphosphate binding region is also proposed for the polyphosphate glucokinase enzyme.

Kinetic mechanism of pyrophosphate-dependent phosphofructokinase from Giardia lamblia.

The steady-state kinetics of the reaction catalyzed by inorganic-pyrophosphate-dependent D-fructose-6-phosphate 1-phosphotransferase from Giardia lamblia have been investigated. The reactants for the forward and reverse reactions were the Mg-chelated complexes of pyrophosphate (PPi) and Pi. Uncomplexed ligands were not substrates. In the direction of phosphorylation of fructose-6-phosphate (F6P), initial velocity double-reciprocal plots for both PPi and F6P were intersecting suggesting sequential addition of substrates. Similarly, intersecting patterns were observed in the reverse reaction with either Pi or fructose-1,6-bisphosphate (FBP) as the variable substrate. Although the catalytic constants for the forward and reverse reactions were found to be identical (83 s-1), the kcat/Km for PPi is about two orders of magnitude higher than the kcat/Km for Pi, indicating that PPi is utilized much more efficiently than Pi. Product inhibition of Pi is competitive vs. PPi and noncompetitive vs. F6P, when the fixed substrate is subsaturating. Product inhibition by FBP was found to be noncompetitive with either Pi or F6P as the variable substrate. These results are consistent with a sequential ordered Bi Bi mechanism with PPi adding first and Pi dissociating last. In the reverse reaction, however, PPi and F6P were found to be noncompetitive with either Pi or FBP. Dead-end inhibition analysis with fructose 2,6-bisphosphate, a competitive substrate analog of FBP, gave uncompetitive inhibition with respect to Pi, indicating that fructose 2,6-bisphosphate (and hence FBP) binds after Pi. This kinetic mechanism is different from that observed with the enzyme from Propionibacterium freudenreichii, Entamoeba histolytica or Mung bean, which were concluded to be rapid equilibrium random mechanism.

Pyrophosphate-dependent phosphofructokinase from Giardia lamblia: purification and characterization.

The pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) from Giardia lamblia has been purified to homogeneity using two methods. The purified enzyme is shown to be homogenous by its migration as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and a single peak on reverse-phase HPLC. The purified enzyme is a monomeric protein of Mr 64 kDa as determined by SDS-PAGE and native PAGE. The enzyme fractionated as a 67-kDa protein by HPLC gel filtration. The pH optima in either the glycolytic or the gluconeogenic direction is near neutral, which is unlike any of the protozoan or bacterial enzymes. The enzyme displayed typical Michaelis-Menten kinetics. The Km values for pyrophosphate and fructose 6-phosphate were 0.039 +/- 0.005 and 0.25 +/- 0.0196 mM, respectively. The initial velocity for the reverse reaction was also found to be hyperbolic. Different nucleoside triphosphates, including ATP, could not substitute for pyrophosphate as the phosphoryl donor. Similar to the pyrophosphate-dependent enzyme from other anaerobic protozoans and bacteria, the Giardial enzyme was not activated by fructose 2,6-bisphosphate, although this metabolite is a competitive inhibitor with respect to fructose 1,6-bisphosphate in the reverse reaction. The pH optima and the molecular weight properties of the Giardial enzyme are different from those of the two classes of the pyrophosphate-dependent phosphofructokinases.

Primary structure of the monomer of the 12S subunit of transcarboxylase as deduced from DNA and characterization of the product expressed in Escherichia coli.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types: a hexameric central 12S subunit to which 6 outer 5S subunits are attached through 12 1.3S biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl coenzyme A and pyruvate serve as substrates to form propionyl coenzyme A (propionyl-CoA) and oxalacetate, the 12S subunit specifically catalyzing one of the two reactions. We report here the cloning, sequencing, and expression of the 12S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 12S peptides with the deduced sequence of an open reading frame present in a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3S biotinyl subunit. The cloned 12S gene encodes a protein of 604 amino acids and of M(r) 65,545. The deduced sequence shows regions of extensive homology with the beta subunit of mammalian propionyl-CoA carboxylase as well as regions of homology with acetyl-CoA carboxylase from several species. Two genomic fragments were subcloned into pUC19 in an orientation such that the 12S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots (immunoblots) which comigrated with authentic 12S. The Escherichia coli-expressed 12S was purified to apparent homogeneity by a three-step procedure and compared with authentic 12S from P. shermanii. Their quaternary structures were identical by electron microscopy, and the E. coli 12S preparation was fully active in the reactions catalyzed by this subunit. We conclude that we have cloned, sequenced, and expressed the 12S subunit which exists in a hexameric active form in E.coli.

Primary structure of the 5 S subunit of transcarboxylase as deduced from the genomic DNA sequence.

Transcarboxylase from Propionibacterium shermanii is a complex biotin-containing enzyme composed of 30 polypeptides of three different types. It is composed of six dimeric outer subunits associated with a central cylindrical hexameric subunit through 12 biotinyl subunits; three outer subunits on each face of the central hexamer. Each outer dimer is termed a 5 S subunit which associates with two biotinyl subunits. The enzyme catalyzes a two-step reaction in which methylmalonyl-CoA and pyruvate form propionyl-CoA and oxalacetate, the 5 S subunit specifically catalyzing one of these reactions. We report here the cloning, sequencing and expression of the monomer of the 5 S subunit. The gene was identified by matching amino acid sequences derived from isolated authentic 5 S peptides with the deduced sequence of an open reading frame present on a cloned P. shermanii genomic fragment known to contain the gene encoding the 1.3 S biotinyl subunit. The cloned 5 S gene encodes a protein of 519 amino acids, M(r) 57,793. The deduced sequence shows regions of extensive homology with that of pyruvate carboxylase and oxalacetate decarboxylase, two enzymes which catalyze the same or reverse reaction. A fragment was subcloned into pUC19 in an orientation such that the 5 S open reading frame could be expressed from the lac promoter of the vector. Crude extracts prepared from these cells contained an immunoreactive band on Western blots which co-migrated with authentic 5 S and were fully active in catalyzing the 5 S partial reaction. We conclude that we have cloned, sequenced and expressed the monomer of the 5 S subunit and that the expressed product is catalytically active.

Determination of endopolyphosphatase using polyphosphate glucokinase.

A method has been developed for determining endopolyphosphatase (polyphosphate depolymerase, EC 3.6.1.10) activity. The enzyme catalyzes the hydrolysis of inorganic polyphosphates [poly(Ps)] by cleaving internal phosphoanhydride bonds without removal of terminal phosphate residues. During the reaction, shorter poly(P) chains are formed and the molar concentration of poly(P) increases. This enzymatic activity is difficult to quantitate, because the substrates and products of the reaction are chemically identical. The commonly used viscometric method lacks sensitivity and cannot be used with shorter poly(P) substrates. The method described here overcomes these problems, and in addition is rapid, simple, and can be used for distinguishing between the endopolyphosphatase and exopolyphosphatase (EC 3.6.1.11) activities. It is based on monitoring the increase in the number of poly(P) chains generated by endopolyphosphatase. For this purpose, the method takes advantage of the specific property of poly(P) glucokinase (EC 2.7.1.63) which utilizes poly(Ps) of different sizes present in the endopolyphosphatase reaction mixture and reduces them to fairly uniform very short-chain product, poly(P)m. The concentration of poly(P)m is expressed in terms of acid-labile phosphorus and is proportional to the duration of the endopolyphosphatase reaction (i.e., the number of original poly(P) chains) and to protein concentration. The increase in the poly(P)m concentration is a relative measure of the endopolyphosphatase activity. Under certain conditions, m equals 3.5 and the activity can be expressed in standard units, since the exact number of poly(P) chains formed by endopolyphosphatase can be calculated from the increase in molar concentration of poly(P)3.5. Accuracy and advantages of the assay are discussed.

Involvement of tryptophan(s) at the active site of polyphosphate/ATP glucokinase from Mycobacterium tuberculosis.

The glucokinase (EC 2.7.1.63) from Mycobacterium tuberculosis catalyzes the phosphorylation of glucose using inorganic polyphosphate (poly(P)) or ATP as the phosphoryl donor. The nature of the poly(P) and ATP sites was investigated by using N-bromosuccinimide (NBS) as a probe for the involvement of tryptophan in substrate binding and/or catalysis. NBS oxidation of the tryptophan(s) resulted in fluorescence quenching with concomitant loss of both the poly(P)- and ATP-dependent glucokinase activities. The inactivation by NBS was not due to extensive structural changes, as evidenced by similar circular dichroism spectra and fluorescence emission maxima for the native and NBS-inactivated enzyme. Both phosphoryl donor substrates in the presence of xylose afforded approximately 65% protection against inactivation by NBS. The Km values of poly(P) and ATP were not altered due to the modification by NBS, while the catalytic efficiency of the enzyme was decreased, suggesting that the essential tryptophan(s) are involved in the catalysis of the substrates. Acrylamide quenching studies indicated that the tryptophan residue(s) were partially shielded by the substrates against quenching. The Stern-Volmer quenching constant (KSV) of the tryptophans in unliganded glucokinase was 3.55 M-1, while KSV values of 2.48 and 2.57 M-1 were obtained in the presence of xylose+poly(P)5 and xylose+ATP, respectively. When the tryptophan-containing peptides were analyzed by peptide mapping, the same peptide was found to be protected by xylose+poly(P)5 and xylose+ATP against oxidation by NBS. The two protected peptides were determined to be identical by N-terminal sequence analysis and amino acid composition.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification of polyphosphate and ATP glucose phosphotransferase from Mycobacterium tuberculosis H37Ra: evidence that poly(P) and ATP glucokinase activities are catalyzed by the same enzyme.

Polyphosphate [poly(P)n]:D-(+)-glucose-6-phosphotransferase (EC 2.7.1.63) from Mycobacterium tuberculosis H37Ra was purified to homogeneity using an improved method which yielded a 634-fold purification with higher recovery. The purified enzyme migrated as a single band with M(r) 33 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The native enzyme was shown to be a dimer by gel filtration using high-performance liquid chromatography (HPLC). The purified enzyme fractionated as a single peak on a C8 reverse-phase HPLC column and was found to display both polyphosphate- and ATP-dependent glucokinase activities. Further evidence that a single protein was responsible for both activities was shown by nondenaturing PAGE, in which the two activities (as determined by an activity stain in dual experiments) were found to comigrate. The C-terminal analysis yielded a single sequence while the N-terminus which was blocked also yielded a single sequence after deblocking. The two activities were found to have the same temperature optimum of 50 degrees C. The pH optima were 9.5 and 8.6-9.5 with poly(P)32 and ATP as the phosphoryl donors, respectively. The apparent Km for poly(P)32 was 18.4 microM while the Km for ATP was 1.46 mM. In addition, the nucleotide analogue, Reactive Blue 4, was found to be a competitive inhibitor with ATP in the ATP-dependent glucokinase reaction, while it displayed noncompetitive inhibition patterns with poly(P) in the poly(P)-dependent glucokinase reaction. It is concluded that the poly(P) and ATP glucokinase activities are catalyzed by the same enzyme but that the two substrates may have different binding sites.

The nonbiotinylated form of the 1.3 s subunit of transcarboxylase binds to avidin (monomeric)-agarose: purification and separation from the biotinylated 1.3 S subunit.

Avidin-biotin technology is used routinely to purify biotin-containing carboxylases and also proteins that have been chemically coupled to biotin. The 1.3 S subunit of transcarboxylase (TC) studied here is the biotin-containing subunit of TC which not only acts as a carboxyl carrier between the CoA ester sites on the central 12 S subunit of TC and keto acid sites on the outer 5 S subunit of TC but also links the 12 S and 5 S subunits together to form a 26 S multisubunit TC complex. The 1.3 S subunit has been cloned, sequenced, and expressed in Escherichia coli. A method for purifying recombinant 1.3 S subunits from E. coli using avidin (monomeric)-agarose column chromatography has been developed. This affinity-purified 1.3 S was found to be homogeneous by SDS-PAGE, amino acid composition, and N-terminal sequence analysis but had a biotin content of only 28% based on moles of biotin per mole of 1.3 S. This lack of stoichiometry was found to be due to copurification of apo-1.3 S as evidenced by the holocarboxylase synthetase reaction. A procedure for separating the apo- and biotinylated 1.3 S forms using hydrophobic interaction chromatography on an Ether 5 PW column is described. The method is based on the difference in hydrophobicity between apo and biotinylated 1.3 S forms. The copurification of apo and biotinylated forms of 1.3 S on the avidin (monomeric)-agarose column was found to be due to specific interaction with avidin rather than to interaction between apo- and biotinylated 1.3 S forms as demonstrated by the fluorescence quenching studies. The results suggest that the avidin-biotin system by itself may not be sufficient to obtain homogeneous biotinyl proteins as nonbiotinyl protein can also bind avidly to such columns.

The polyphosphate- and ATP-dependent glucokinase from Propionibacterium shermanii: both activities are catalyzed by the same protein.

The glucokinase (EC 2.7.1.63) from Propionibacterium shermanii phosphorylates glucose using inorganic polyphosphate (poly(P)) or ATP as the phosphate donor. In this investigation, we have purified the glucokinase to homogeneity, using two methods and show that the polyphosphate and ATP-dependent glucokinase activities eluted as a single protein. The protein peak is shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse phase HPLC, and N-terminal sequence analysis. The purified protein eluted as a single peak from gel filtration and hydrophobic interaction HPLC columns and was found to display both the poly(P) and ATP glucokinase activities. Likewise, the two activities comigrated on a native isoelectric focusing gel. In addition, two analogues of ATP with different reactive groups displayed different inhibition patterns with respect to ATP and poly(P). The 2',3'-dialdehyde of ATP, whose reactive group is the dialdehyde of the ribose ring, showed competitive and noncompetitive patterns with respect to ATP and poly(P), respectively. While, 5'-p-fluorosulfonylbenzoyl adenosine, whose reactive sulfonyl fluoride group is related to the gamma-phosphoryl group of ATP, displayed competitive inhibition patterns with both ATP and poly(P). These observations provide evidence that the polyphosphate and ATP-dependent glucokinase activities of P. shermanii are the catalytic properties of a single enzyme and that the two substrates may have different binding sites on the enzyme with a common phosphorylating center.

Isolation of a carboxyphosphate intermediate and the locus of acetyl-CoA action in the pyruvate carboxylase reaction.

When chicken liver pyruvate carboxylase was incubated with either H14CO3- or gamma-[32P]ATP, a labeled carboxyphospho-enzyme intermediate could be isolated. The complex was catalytically competent, as determined by its subsequent ability to transfer either 14CO2 to pyruvate or 32P to ADP. While the carboxyphospho-enzyme complex was inherently unstable and the stoichiometry of the transfer was variable depending on experimental conditions, both the [14C]carboxyphospho-enzyme and the carboxy[32P]phospho-enzyme had similar half-lives. Acetyl-CoA was shown to be involved in the conversion of the carboxyphospho-enzyme complex to the more stable carboxybiotin-enzyme species, which was consistent with the effects of acetyl-CoA on isotope exchange reactions involving ATP. We were unable to detect the formation of a phosphorylated biotin derivative during the ATP cleavage reaction. In the presence of K+ and at pH 9.5, the acetyl-CoA-independent activity of chicken liver pyruvate carboxylase approached 2% of the acetyl-CoA-stimulated rate, which represents a 30-fold increase on previously reported activity for this enzyme.

Structural and functional analysis of the human neutrophil 1-15 antigen, an Mr 65,000 to 70,000 activation-associated membrane proteinase.

Previous studies with the anti-neutrophil/antichymotrypsin mAb 1-15 have identified an activation-associated, chymotrypsin-like activity within the membrane fraction of isolated human neutrophils (PMN). In the present study, the molecular and biochemical characteristics of mAb 1-15 Ag/proteinase were determined. On casein/acrylamide sizing gels, PMN membrane preparations were found to contain an Mr 58,000 to 84,000 band of Ca2(+)-dependent proteinase activity. Reducing and nonreducing SDS-PAGE of mAb 1-15-affinity-purified membrane proteins demonstrated specific recovery of an enzymatically active Mr 65,000 to 70,000 chymotrypsin-like Ag. The presence of a distinct membrane serine esterase of isoelectric point 6.3/Mr 65,000 to 70,000 was confirmed in active site-labeling experiments with the serine proteinase inhibitor [3H]diisopropylfluorophosphate (DFP). Substrate-affinity chromatography with phe-Sepharose or FMLP-Sepharose provided partial purification of enzyme activity among Mr 65,000 to 70,000 FMLP- or phe-binding proteins. Enzyme inhibition was obtained by incubation with mAb 1-15, DFP, N-carbobenzoxyl-phe-chlormethyl ketone, or PMSF, but not tosyl-amide-phenylethylchlormethyl ketone, bestatin, aprotinin, or phosphoramidon. In HPLC analysis, [3H]DFP labeled proteinase was found to comigrate with one of three FMLP-affinity-labeled membrane peaks, but unlike the FMLP surface receptor the DFP-labeling membrane proteinase was not modified by endoglycosidase F. We conclude that the mAb 1-15 Ag, which appears to play a role in PMN activation, is a distinct, active, Mr 65,000 to 70,000 serine proteinase with affinity for substrate sites containing aromatic amino acids.

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

Involvement and identification of a tryptophanyl residue at the pyruvate binding site of transcarboxylase.

Transcarboxylase (TC) from Propionibacterium shermanii consists of a central hexameric 12S subunit to which 6 outer dimeric 5S subunits are attached through 12 biotinyl 1.3S subunits. The enzyme catalyzes the transfer of a carboxyl group from methylmalonyl-CoA to pyruvate, forming oxalacetate and propionyl-CoA. The pyruvate binding site, located on the 5S subunit, was examined by monitoring the intrinsic fluorescence quenching accompanying the incremental addition of pyruvate to either TC or the 5S subunit. The quenching studies indicate that there are two binding sites for pyruvate with apparent dissociation constants of 0.23 and 1.25 mM for intact TC and of 0.18 and 1.20 mM for the outer 5S subunit. The microenvironment of the Trp(s) sensitive to pyruvate binding was analyzed by using the neutral quencher acrylamide. With TC, the fractional accessible fluorescence (fa) was 0.64, whereas a fa value of 0.56 was obtained in the presence of pyruvate. A 27% decrease in fa was observed with the outer 5S subunit in the presence of pyruvate as compared to the free 5S subunit. By labeling the outer subunit in the absence of pyruvate with 2,4-dinitrophenylsulfenyl chloride (DNPS-Cl), a tryptic peptide containing DNPS-labeled Trp was isolated; the sequence was determined and identified with the amino-terminal residues 67-75 of the outer subunit that has been derived from DNA-sequencing studies. Trp-73 contained the DNPS label; its labeling was inhibited by pyruvate. A sequence comparison with other biotinyl enzymes shows that the sequence 67-75 is highly conserved.(ABSTRACT TRUNCATED AT 250 WORDS)

The ATP/AMP binding site of pyruvate,phosphate dikinase: selective modification with fluorescein isothiocyanate.

Pyruvate,phosphate dikinase from Propionibacterium shermanii is strongly inhibited by fluorescein 5'-isothiocyanate (FITC). The time course of inactivation is biphasic, but the dependence of the pseudo-first-order rate constants on the inhibitor concentration indicates the formation of a reversible complex with the enzyme prior to covalent modification. The substrate/product nucleotide pairs MgATP and MgAMP protected against inactivation, while in the absence of Mg2+, both the nucleotides were ineffective. Previously, an essential lysine at the ATP/AMP subsite of the enzyme from Bacteroides symbiosus had been implicated by use of the 2',3'-dialdehyde of AMP (oAMP) [Evans, C. T., Goss, N. H., & Wood, H. G. (1980) Biochemistry 19, 5809]. The inhibition by FITC was competitive with MgAMP, and a multiple inhibition analysis plot indicated that binding of oAMP and FITC was mutually exclusive. These observations suggest that FITC and oAMP bind at the nucleotide binding site and probably to the same reactive lysine that is modified by oAMP. With peptide mapping by high-performance liquid chromatography, FITC was found to be a suitable probe for isolating the peptide from the ATP/AMP subsite.