Purine nucleoside phosphorylase from Escherichia coli and Salmonella typhimurium. Purification and some properties.
The purine nucleoside phosphorylases from Escherichia coli and from Salmonella typhimurium have been purified to electrophoretic homogeneity and crystallized. Comparative studies revealed that the two enzymes are very much alike. They obey simple Michaelis-Menten kinetics for their substrates with the exception of phosphate for which they show negative cooperativity. Gel filtration on Sephadex G-200 of the native enzymes revealed a molecular weight for both enzymes of 138000 plus or minus 10%. By use of dodecylsulphate gel electrophoresis a subunit molecular weight of 23700 plus or minus 5% was determined, suggesting that both enzymes consist of six subunits of equal molecular weight. When the subunits were partially crosslinked with dimethyl suberimidate before dodecylsulphate electrophoresis six protein bands were observed in agreement with the proposed oligomeric state of the enzyme, consisting of six subunits of equal molecular weight. Analysis of the amino acid composition also indicates that the subunits are identical. 6M guanidinium chloride dissociates the enzymes; association experiments with native and succinylated enzymes suggested that only the hexameric form is active. Both enzymes could be dissociated into subunits by p-chloromercuribenzoate; this dissociation is prevented by the substrates: the nucleosides, the pentose 1-phosphates, and mixtures of phosphate and purine bases.
D000596:Amino Acids / Q000032:analysis; D003460:Crystallization; D004120:Dimethyl Suberimidate; D004355:Drug Stability; D004789:Enzyme Activation; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D010430:Pentosyltransferases / Q000302:isolation & purification*; D011683:Purine-Nucleoside Phosphorylase / Q000302:isolation & purification* / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D046911:Macromolecular Substances; D004120:Dimethyl Suberimidate; D010430:Pentosyltransferases; D011683:Purine-Nucleoside Phosphorylase