Biochimica et Biophysica Acta (BBA) - Enzymology最新文献

The role of exposed tyrosine side-chains in enzyme-catalyzed reaction by sweet almond emulsion β-d-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) has been studied using N-acetylimidazole as the specific reagent. The changes in activity, binding affinity and kinetic parameters (K_m, V) as a result of acetylation of the phenolic hydroxyl groups have been determined. The acetylation increased the K_m values of both β-glucosidase and β-galactosidase activities, whereas V remained unchanged. Similarly, the binding affinity for immobilized phenyl β-d-glucopyranoside decreased appreciably. After the removal of the acetyl groups the enzyme regained 96% of the original activity. It is concluded that the tyrosine moieties, located in the active centre of the enzyme, have both glucoside and galactoside binding functions.

Modification of ribulosebisphosphate carboxylase (3-phospho-d-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) by diethylpyrocarbonate or rose bengal-sensitized photooxidation caused rapid inactivation of the enzyme. The photooxidation proceeded following pseudo-first-order reaction kinetics showing a maximal value at pH 8.0. The fully activated enzyme was more sensitive to photooxidation as compared to the unactivated enzyme. The enzyme partially inactivated by photooxidation was fully sensitive to the positive effectors. The photooxidised enzyme showed a characteristic increase in absorbance at 250 nm which was dependent on the extent of inactivation. The kinetic analyses and correlation of the spectral changes with the activity indicated that the inactivation by diethylpyrocarbonate resulted from the modification of an average one histidine residue/70 000-dalton combination of large and small subunit. Sulfhydryl, lysine and tyrosine residues were not modified by diethylpyrocarbonate treatment. Ribulosebisphosphate and some effectors of the enzyme offered significant protection against diethylpyrocarbonate modification indicating that diethylpyrocarbonate was interacting with the enzyme at or near the active site.

We described the partial characterization and some properties of fibroblast and leucocyte neuraminidase towards 2 → 3 and 2 → 6 sialyllactose, and 2 → 3 and 2 → 6 sialylhexasaccharide which were isolated from the urine of a patient with adult sialidosis with partial β-galactosidase deficiency. Neuraminidase activities were assayed using the radioactive-labeled derivatives of these saccharide substrates. These neuraminidases (acylneuraminyl hydrolase, EC 3.2.1.18) were partially inactivated by homogenization, sonication and freeze-thawing treatment. The leucocyte neuraminidase was more labile than that of fibroblasts. Fibroblast neuraminidase had about a 10-fold higher activity than leucocyte neuraminidase towards the respective substrates. The neuraminidase from fibroblasts and leucocytes were each able to hydrolyze 2 → 3 isomers 2–3-times faster than 2 → 6 isomers and the sialyllactoses 1.5–3.0-times faster than sialylhexasaccharides. Neuraminidase activities towards all four substrates were deficient in fibroblasts and leucocytes from the patients with adult sialidosis. Loss of activity was especially prominent in fibroblasts, while considerable residual activities (about 20–30%) remained in leucocytes. In mucolipidosis II and III patients, these neuraminidase activities showed normal levels in leucocytes, although they were decreased in fibroblasts. The discrepancy between neuraminidase activities towards 2 → 3 and 2 → 6 isomers was not found in all the cases.

Phosphorylation of fructose-bisphosphatase (d-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) by the catalytic subunit of cyclic AMP-dependent protein kinase from pig muscle decreased the K_0.5 for fructose-bisphosphate from 21 to 11 μM. When the phosphorylated fructose-bisphosphatase was treated with trypsin the K_0.5 increased to 22 μM. The K_0.5 also increased when the phosphoenzyme was treated with a partially purified phosphatase from rat liver. There was no difference between the unphosphorylated and phosphorylated enzyme with respect to pH dependence, the pH optimum being about 7.0 for both. Limited treatment of fructose-bisphosphatase with subtilisin, which cleaves the enzyme at its unphosphorylatable N-terminal part, increased the pH optimum more than limited treatment with trypsin, which releases the phosphorylated peptide at the C-terminal part of fructose-bisphosphatase. The phosphorylated site on the phosphorylated fructose-bisphosphatase was more easily split off by trypsin treatment than the corresponding unphosphorylated site. The results suggest in addition to the glucagon-induced phosphorylation of fructose-bisphosphatase described by Claus et al. [1] that the phosphorylation-dephosphorylation of fructose-bisphosphatase could be of importance for the hormonal regulation of the enzyme in vivo.

An α-l-fucosidase (α-l-fucoside fucohydrolase, EC 3.2.1.51) has been isolated from porcine thyroid tissue and purified 10 800-fold using a combination of ion exchange, affinity and molecular sieve chromatography. The enzyme appears homogeneous by SDS electrophoresis but isoelectric focusing procedures detect considerable heterogeneity. The enzyme is a glycoprotein and this fact interferes with accurate molecular weight estimates by SDS electrophoresis or molecular sieve techniques. The enzyme appears, however, to be a tetramer and density gradient measurements set its molecular weight at 19200 ± 3000. The enzyme exhibits an optimum at a pH of 5.1 and shows a high order of specificity for l-fucose units linked through α bonds. Both sulfhydryl and carboxyl groups appear necessary for enzyme activity. The enzyme does not attack intact thyroglobulin directly but will remove fucosyl residues from the glycome moiety if the protein portion is largely removed. The enzyme thus functions in a salvage role as thyroglobulin is degraded.

We have developed a mathematical model of the nonideal case in which enzymatic activity changes may also result from modification of non-essential groups. As an illustration of this method, the number of essential carboxyl groups in pig heart fumarase (l-malate hydro-lyase, EC 4.2.1.2) was determined by the differential labeling technique. Enzymatic activity was related to the number of modified carboxyl groups according to the model and the results were compatible with the existence of two essential carboxyl groups in fumarase.

The 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5′-phosphate deaminase was partially purified from cell extracts of Candida guilliermondii ATCC 9058. The enzyme requires Mg²⁺ for activity. Maximal activity was observed at pH 7.3. The enzyme converts its substrate, 2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5′-phosphate, to 2,5-diamino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-phosphate. This labile compound was treated with diacetyl and the resulting 6,7-dimethyl-8-ribityllumazine 5′-phosphate was identified by comparison with a synthetic sample.

The steady-state kinetics of rat liver hexose-6-phosphate dehydrogenase (β-d-glucose: NAD(P)⁺ 1-oxidoreductase, EC 1.1.1.47) using glucose 6-phosphate and NADP⁺ as substrates is studied. NADPH has been found to inhibit the enzyme noncompetitively with respect to NADP⁺, and uncompetitively with respect to glucose 6-phosphate. At a given concentration of glucose 6-phosphate, the reaction follows the basic inhibition equation. This suggests the presence of the enzyme-NADP⁺-NADPH complex, and contrasts with the NADPH inhibition of glucose-6-phosphate dehydrogenase which is competitive with respect to NADP⁺. An attempt was made to estimate the in vivo activities of the two enzymes in rat liver in the presence of NADPH at various NADPH/NADP⁺ ratios. The results show that the two enzymes appear to be at about the same level of activity in normal rat liver where the coenzyme redox ratio is 110 and the glucose 6-phosphate concentration is 217 μM. Under the same conditions, but with 50 μM dehydroepiandrosterone, a potent inhibitor of glucose-6-phosphate dehydrogenase, but not of hexose-6-phosphate dehydrogenase, the latter enzyme is estimated to be 1.6-times as active as the former. Such differential effects of NADPH and steroids on the two enzymes may support our notion that hexose-6-phosphate dehydrogenase may have advantages over glucose-6-phosphate dehydrogenase (d-glucose-6-phosphate: NADP⁺ 1-oxidoreductase, EC 1.1.1.49) in steroid-metabolizing tissues (the activity of hexose-6-phosphate dehydrogenase is not, or less, affected by steroids or NADPH).

The degradation of des-Arg⁹-bradykinin and its analogues by highly purified preparations of hog lung and kidney kininase II (angiotensin-converting enzyme; peptidyldipeptide hydrolase, EC 3.4.15.1) was studied. The degradative peptide fragments were separated and isolated by high performance liquid chromatography and identified by amino acid analysis. Both enzymes released C-terminal tripeptides from des-Arg⁹-bradykinin, des-Arg⁹-(Leu⁸)-bradykinin, Pro-Pro-Gly-Phe-Ser-Pro-Phe, Pro-Gly-Phe-Ser-Pro-Phe, Gly-Phe-Ser-Pro-Phe, Bz-Gly-Ser-Pro-Phe and Bz-Gly-Ala-Pro-Phe. Hydrolysis of Phe-Ser-Pro-Phe, Bz-Gly-His-Pro-Phe, Bz-Gly-Phe-Pro-Phe and Bz-Gly-Gly-Pro-Phe by both enzymes was negligible. These data indicate that kininase II can release C-terminal tripeptides of substrates having a proline residue in the penultimate position such as des-Arg⁹-bradykinin and its analogues, and that this enzyme is able not only to act as a dipeptidyl carboxypeptidase but also acts as a tripeptidyl carboxypeptidase. The tripeptidyl carboxypeptidase activity of this enzyme was sensitive to inhibition by kininase II inhibitors.