scholarly journals A stereochemical investigation of the hydrolysis of cyclic AMP and the (Sp)-and (Rp)-diastereoisomers of adenosine cyclic 3′:5′-phosphorothioate by bovine heart and baker's-yeast cyclic AMP phosphodiesterases

1982 ◽  
Vol 203 (2) ◽  
pp. 461-470 ◽  
Author(s):  
R L Jarvest ◽  
G Lowe ◽  
J Baraniak ◽  
W J Stec
Keyword(s):  
Cyclic Amp ◽  
Phosphorus Atom ◽  
Metal Ion ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Bivalent Metal ◽  
Cyclic Amp Phosphodiesterase ◽  
Bovine Heart ◽  
Hydrolysis Of

Bovine heart cyclic AMP phosphodiesterase, which has a requirement for Mg2+, hydrolyses cyclic AMP with inversion of configuration at the phosphorus atom, but only the (Sp)-diastereoisomer of adenosine cyclic 3′:5′-phosphorothioate is hydrolysed by this enzyme. By contrast, the low-affinity yeast cyclic AMP phosphodiesterase, which contains tightly bound Zn2+, hydrolyses both the (Sp)- and the (Rp)-diastereoisomers of adenosine cyclic 3′:5′-phosphorothioate, the (Rp)-diastereoisomer being the preferred substrate under V max. conditions. Both of the diastereoisomers of adenosine cyclic 3′:5′-phosphorothioate, as well as cyclic AMP, are hydrolysed with inversion of configuration at the phosphorus atom by the yeast enzyme. It is proposed that, with both enzymes, the bivalent metal ion co-ordinates with the phosphate residue of the substrate, and that hydrolysis is catalysed by a direct ‘in-line’ mechanism.

scholarly journals The specificity of yeast low-Km cyclic AMP phosphodiesterase towards free bivalent metal ions and the diastereoisomers of cyclic adenosine phosphorothioate

1985 ◽  
Vol 226 (3) ◽  
pp. 897-900 ◽  
Author(s):  
K Suoranta ◽  
J Londesborough
Keyword(s):  
Cyclic Amp ◽  
Metal Ions ◽  
Metal Ion ◽  
Relative Activity ◽  
Bivalent Metal ◽  
Cyclic Amp Phosphodiesterase ◽  
Bivalent Metal Ions

The relative activity of a zinc-containing cyclic AMP phosphodiesterase towards the (Sp)- compared with the (Rp)-diastereoisomer of cyclic adensine phosphorothioate varied with the identity of the free bivalent metal ion from more than 35 to 0.074 along the series Mg2+ greater than Mn2+ greater than Co2+ greater than Zn2+ greater than Cd2+, showing that this ion, and not the tightly bound zinc, bonds to the phosphorothioate moiety of the substrate.

scholarly journals Solubilization and other studies on adenylate cyclase of baker's yeast

1976 ◽  
Vol 159 (2) ◽  
pp. 363-370 ◽  
Author(s):  
K Varimo ◽  
J Londesborough
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cyclic Amp ◽  
Adenylate Cyclase ◽  
Gel Filtration ◽  
Baker’S Yeast ◽  
Baker's Yeast ◽  
Centrifugal Forces ◽  
Wide Range ◽  
Solubilized Enzyme ◽  
Fold Activation

1. Adenylate cyclase of Saccharomyces cerevisiae was sedimented from mechanically disintegrated preparations of yeast over an unusually wide range of centrifugal forces. 2. The enzyme was readily solubilized by Ficoll and by Lubrol PX. Lubrol caused a 2-fold activation. 3. Both particle-bound and Lubrol-solubilized enzyme had an apparent Km for ATP of 1.6 mM in the presence of 0.4 mM-cyclic AMP and 5 mM-MnCl2 at pH 6.2 and 30°C. 4. The Lubrol-solubilized enzyme behaved on gel filtration as a monodisperse protein with an apparent mol.wt. of about 450000.

Purification and properties of three endopeptidases from baker's yeast

1989 ◽  
Vol 35 (2) ◽  
pp. 295-303 ◽  
Author(s):  
Jerzy Nowak ◽  
Hsin Tsai
Keyword(s):  
Ethyl Ester ◽  
Baker’S Yeast ◽  
Molecular Forms ◽  
Acid Proteinase ◽  
Baker's Yeast ◽  
Ph Optimum ◽  
Protein Substrates ◽  
Milk Clotting ◽  
Hydrolysis Of ◽  
Milk Clotting Activity

Three endopeptidases, proteinases A, B, and Y, were purified from baker's yeast, Saccharomyces cerevisiae. Two molecular forms of proteinase A (PRA), Mr 45 000 and 54 000, (estimated on SDS-PAGE) were obtained. Both forms were inhibited by pepstatin and other acid proteinase inhibitors. The enzyme digested hemoglobin most rapidly at pH 2.7–3.2 and casein at pH 2.4–2.8 and 5.5–6.0. The optimum pH for hydrolysis of protein substrates could be shifted to about 5 with 4–6 M urea. Urea also stimulated the enzyme activity by 30–50%. As other acid proteinases, the enzyme preferentially cleaved peptide bonds of X–Tyr and X–Phe type. A proteinase B (PRB) preparation of approximately Mr 33 000 possessed milk clotting activity and showed an inhibition pattern typical for seryl-sulfhydryl proteases. The purified enzyme could be stabilized with 40% glycerol and stored at −20 °C without significant loss of activity for several months. The third endopeptidase, designated PRY, of Mr 72 000 when estimated by Sephadex G-100 gel filtration, had properties resembling PRA and PRB. Similar to PRB, it could be inhibited by up to 90% with phenylmethylsulfonyl fluoride and para-chloromercuribenzoate and preferentially hydrolyzed the Leu15–Tyr16 peptide bond of the oxidized β-chain of insulin. On the other hand, contrary to PRB, it had neither milk clotting activity nor esterolytic activity toward N-acetyl-L-tyrosine ethyl ester and N-benzoyl-L-tyrosine ethyl ester and was stable during storage at −20 °C without glycerol. The enzyme also showed a lower pH optimum for hydrolysis of casein yellow than PRB. Similar to PRA, 4 M urea shifted its pH optimum for hydrolysis of protein substrates. PRY degraded apo-aminopeptidase Y much more efficiently than PRB or a PRA–PRB mixture. The possibility of PRY being a precursor form of PRA and PRB is discussed.Key words: yeast, endopeptidase, proteinase, purification.

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