Orestes Tsolas

Modification of fructose bisphosphatase by a proteolytic enzyme from rat liver lysosomes

Membrane preparations derived from the lysosome-rich heavy particle fraction from rat liver contain a proteinase (converting enzyme) that increases the activity of rabbit liver fructose 1,6-bisphosphatase, assayed at pH 9.2, by approximately six-fold. The activity assayed at pH 7.5 is slightly decreased. These changes are associated with the hydrolysis of several peptide bonds in the tetrameric enzyme (subunit molecular weight =36,000). Initially, hydrolysis appears to occur at either of two distinctly different peptide bonds, yielding modified subunits having molecular weights of approximately 29,000 and 26,000, respectively. The larger modified subunit is resistant to further degradation, but the 26,000 subunit is hydrolyzed to fragments whose mass is approximately 13,000 daltons. The cleaved protein retains its original size and tetrameric structure, but is dissociated into its component peptides on treatment with sodium dodecyl sulfate or during carboxymethylation in urea. One of the sites of hydrolysis has been identified by sequence analysis as the peptide bond between residues Asn 64 and Val 65 ; cleavage of this bond yields the 29,000 subunit and a 6,700 dalton peptide containing the NH 2 -terminus. This bond is adjacent to one of the sites of cleavage by subtilisin reported earlier, supporting the view that in the tetrameric protein each subunit possesses an exposed peptide region that is susceptible to proteolytic attack.

Properties of a fructose 1,6-Bisphosphatase converting enzyme in rat liver lysosomes

Abstract An enzyme present in rat liver lysosomes catalyzes the conversion of neutral rabbit liver fructose 1,6-bisphosphatase (Fru-P 2 ase, EC 3.1.3.11) to a form having maximum activity at pH 9.2. The converting enzyme is partly released when lysosomes are subjected to a single freeze-thaw cycle, but a significant fraction tends to remain with the lysosomal membrane fraction even after repeated freezing and thawing. After repeated freezing and thawing hexosaminidase and cathepsin D are also partly membrane-bound, but cathepsins A, B, and C are completely solubilized. The membrane-bound enzymes, unlike those in intact lysosomes, are not cryptic. The converting enzyme activity is inactivated by phenylmethanesulfonyl fluoride, and is almost completely inactive after exposure to iodoacetic acid or tosylamido-2-phenylethyl and N -α-tosyl lysyl chloromethyl ketones. Unlike cathepsin B, it is not inhibited by leupeptin. Converting enzyme is unstable above pH 6.5, and this property also serves to distinguish it from cathepsins B and D. The results suggest that the converting enzyme is not identical to any of the well-characterized cathepsins.

Rabbit liver fructose 1,6-bisphosphatase: The sequence of the amino-terminal region

Abstract A cyanogen bromide peptide, isolated from the NH 2 -terminus of rabbit liver FruP 2 ase, has been shown to have the following structure: Acetyl-Ala-Asp-Lys-Ala-Pro-Phe-Asp-Thr-Asp-Ile-Ser-Thr-Met-Thr-Arg-Phe-Val-Met. Previous evidence that the enzyme contains tryptophan located near the NH 2 -terminus and also that this tryptophan is lost on exposure of the enzyme to lysosomal fractions must be reevaluated. It is unlikely that the NH 2 -terminus would be reacetylated following proteolytic modification of this portion of the molecule.

Rabbit liver fructose-1,6-bisphosphatase: Location of an active site lysyl residue in the COOH-terminal fragment generated by a lysosomal proteinase

Abstract Digestion of native rabbit liver fructose-1,6-bisphosphatase (Fru-P 2 ase, EC 3.1.3.11) with a membrane-bound proteinase from rat liver lysosomes yields a fragment of M r = 9850. This peptide contains the COOH terminus of the Fru-P 2 ase polypeptide chain and also the cyanogen bromide peptide (BrCN5) carrying the active site lysyl residue. The sequence of BrCN5 and its location with respect to the COOH terminus of the polypeptide chain have been determined. The active site lysyl residue is located at approximately residue −54 from the COOH terminus. The bond hydrolyzed by the lysosomal proteinase is located between residues −88 and −89 from the COOH terminus.

Crystalline fructose 1,6-bisphosphatase from chicken breast muscle

Abstract Fructose 1,6-bisphosphatase has been isolated and crystallized in high yield from chicken breast muscle, which is a rich source of this enzyme. The specific activity assayed at pH 7.4 and 25 °C in the presence of 0.2 m m MnCl 2 0.1 m m EDTA, and 40 m m ammonium sulfate is 50–60 units/mg, making this one of the most active fructose bisphosphatases yet described. The K m for fructose bisphosphate is 8.3 μ m . AMP (0.4 μ m ) inhibits the activity at pH 7.4 almost completely. EDTA can be replaced as activator by citrate or histidine, which both give maximum activation at millimolar concentrations. Citrate is as effective as EDTA. The enzyme has a molecular weight of 144,000 and is composed of four subunits having a molecular weight of 36,000. Amino- and carboxy-terminal analyses indicate that the subunits are identical.

Isolation of a peptide containing a histidinyl-cysteinyl sequence from the active center of transaldolase

Abstract Transaldolase isozyme III contains one histidine and two cysteine residues per subunit, both of which have been implicated in the mechanism of action of the enzyme. A nonapeptide containing the single histidine has now been isolated and its sequence has been shown to be Tyr-Gly-Ile-His-Cys-Asn-Lhr-Leu-Leu, containing a histidinyl-cysteinyl sequence. The proximity of the nitrogen of the imidazole group and the sulphur atom suggests that interaction of these two side chains may play a role in the dealdolization reaction. A tentative mechanism is proposed.

Purification and crystallization of transaldolase isozyme I and evidence for different genetic origin of isozymes I and III in Candida utilis

Abstract A modified procedure for the purification and crystallization of isozymes I and III of transaldolase from extracts of Candida utilis has been developed which makes both enzymes available in sufficient quantity for structural studies. Each is composed of a pair of identical subunits, but the molecular weight of isozyme I is somewhat larger than that of isozyme III. An important difference is in the number of histidine residues: one per subunit in isozyme III and two per subunit in isozyme I. A nonapeptide containing both histidine residues has now been isolated from isozyme I; its sequence is identical to that of the corresponding segment from isozyme III, except that tyrosine is replaced by histidine: His (in place of Tyr)-Gly-Ile-His-Cys-Asx-Thr-Leu-Leu. This amino acid substitution establishes that two different genes code for the two isozymes.

Half-of-the-sites reactivity and all-of-the-sites substrate binding in transaldolase

Abstract Transaldolase from Candida utilis is a dimeric protein composed of two identical subunits. The cleavage of fructose 6-phosphate by this enzyme was followed in a rapidmixing spectrophotometer. A very rapid reaction was observed during which 1 mol of glyceraldehyde 3-phosphate/mol of enzyme was released, followed by a much slower reaction in which additional glyceraldehyde 3-phosphate was formed. Binding studies carried out with the same substrate showed that two equivalents of dihydroxyacetone were bound. These results indicate that both sites are active, but that only one functions in the rapid catalytic reaction. The half-of-the-sites reactivity of transaldolase may be attributed to a high degree of negative cooperativity between the two subunits.

Half-of-the-sites activity of transaldolase: Titration of the active site and characteristics of the slow reaction catalyzed by the second subunit

Abstract When transaldolase is incubated with the donor substrate fructose 6-phosphate in the absence of the acceptor substrate erythrose 4-phosphate, there is a very rapid release of glyceraldehyde 3-phosphate, approaching a ratio of 1 mol/mol of enzyme within 1 min and corresponding to the formation of 1 mol of dihydroxyacetone-enzyme intermediate. This burst reaction is proportional to the amount of enzyme present and takes place in the pH range of the enzymatic activity. It is inhibited by phosphate, which is a competitive inhibitor of fructose 6-phosphate in the enzymatic transfer reaction. Following the burst reaction, there is a slow reaction of the enzyme with fructose 6-phosphate with a kcat 1000 times less than that of the complete reaction. This slow reaction, but not the burst, is also observed with the reduced dihydroxyacetone-transaldolase intermediate, which is inactive in the complete reaction. The Km for fructose 6-phosphate is in the same range for both reactions. Transaldolase is a dimer composed of identical subunits, and the data indicate that it is a half-site enzyme exhibiting almost complete negative cooperativity. If one active site is blocked by reduction of the Schiffs base intermediate, the second site still catalyzes the slow cleavage of fructose 6-phosphate. The rate of this slow reaction reflects the extent of the negative cooperativity.

PARTIAL AMINO ACID SEQUENCE OF RABBIT LIVER FRUCTOSE 1,6-BISPHOSPHATASE (Fru-P2ase, EC 3.1.3.11) AND SITES OF CLEAVAGE BY PROTEINASES

ABSTRACT All vertebrate fructose 1,6-bisphosphatases studied were found to be susceptible to limited proteolysis by subtilisin or by an endogenous lysosomal proteinase. The amino acid sequence adjacent to the proteinase-sensitive region appears to be highly conserved, suggesting that proteolysis may play a critical role in the function of the enzyme. One possible function is release from inhibition by AMP. At pH 5.5 endogenous modification appears to be due to a specific lysosomal proteinase, which is distinct from cathepsins A, B, C or D. Susceptibility of rabbit liver Fru-P2ase to subtilisin can be employed to monitor ligand-induced changes in the conformation of the protein. This technique has provided evidence for an interaction between rabbit liver Fru-P2ase and rabbit liver aldolase, enzymes that catalyze successive steps in gluconeogenesis. The interaction is tissue-specific, and is not seen when either enzyme from liver is replaced by its muscle counterpart.