Chemical Modification and Kinetic Studies on Glucarate Hydro‐Lyase from a Species of Pseudomonas A

Abstract

Glucarate hydro‐lyase, obtained from cells of Pseudomonas A grown on glucarate as the sole source of carbon, was purified 40‐fold in 70% yield. The effects of a variety of chemical modifiers and competitive inhibitors on the enzyme activity were studied. Enzyme activity was irreversibly lost by incubating the enzyme with p‐chloromercuribenzene sulphonate. (+)‐Tartrate, a competitive inhibitor of the enzyme, protected the enzyme from inhibition by this reagent but was not effective when the enzyme was inhibited by coupling it to a diazonium salt. Glucarate, unlike (+)‐tartrate, was an effective protecting group against this type of inhibition. The roles of the modified amino acid residues are discussed.

Studies on glucarate catabolism: the oxodeoxyglucarate aldolase activity of glucarate hydrolyase from Pseudomonas acidovorans (Short Communication)

“…10013 was obtained from the National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, U.K. The organism was maintained and grown under conditions identical with those described by Jeffcoat (1972). Protein was determined with reference to bovine serum albumin (BDH Biochemicals, Poole, Dorset, U.K.) from a calibration curve prepared by the method of Lowry et al (1951).…”

“…Glucarate hydrolyase from Ps. acidovorans was assayed as described by Jeffcoat (1972). 4-Deoxy-5-oxoglucarate aldolase activity was measured by a coupled assay with 10 units of pig heart lactate dehydrogenase [Boehringer Corp. (London) Ltd., London W.5, U.K.], 0.5,umol of NADH, 1,umol of oxodeoxyglucarate, 501umol…”

“…Glucarate hydro-lyase purified from Ps. acidovorans as described by Jeffcoat (1972) was further purified by gel filtration on a column (100cm x 2cm) of Bio-Gel P-200 (50-100 mesh), equilibrated with 0.05M-potassium phosphate buffer-0.1 M-NaCl, pH7.5. These conditions were also used for gel filtration on Sephadex G-100.…”

“…Preparation of enzyme substrates. Potassium hydrogen glucarate and oxodeoxyglucarate were prepared as described by Jeffcoat (1972).…”

Studies on the subunit structure of 4-deoxy-5-oxoglucarate hydro-lyase (decarboxylating) from Pseudomonas acidovorans

“…Until recently little evidence was available to suggest that any other enzyme could be classified in this way, but there is now increasing support that such similarities also exist between hydro-lyases isolated from different sources. This stems from the isolation of Mg2+dependent hydro-lyases (Blumenthal & Fish, 1963;Jeffcoat, 1972) and Schiff-base-forming hydro-lyases (Portsmouth et al, 1967;Jeffcoat et al, 1969). Closer links between aldolases and hydro-lyases are now emerging, since preliminary observations have suggested that metal-ion-dependent aldolases (Morse & Horecker, 1968) and Mg2+-dependent hydrolyases (Jeffcoat, 1972) utilize the formation of a metal-containing ring structure in the enzymesubstrate complex, and Jeffcoat (1974) has demonstrated the Mg2+-dependent aldol cleavage of 4deoxy-5-oxoglucarate by glucarate hydro-lyase.…”