Purification and properties of agmatine ureohydrolyase, a putrescine biosynthetic enzyme in Escherichia coli.
The putrescine biosynthetic enzyme agmatine ureohydrolase (AUH) (EC 3.5.3.11) catalyzes the conversion of agmatine to putrescine in Escherichia coli. AUH was purified approximately 1,600-fold from an E. coli strain transformed with the plasmid pKA5 bearing the speB gene encoding the enzyme. The purification procedure included ammonium sulfate precipitation, heat treatment, and DEAE-sephacel column chromatography. The molecular mass of nondenatured AUH is approximately 80,000 daltons as determined by gel-sieving column chromatography, while on denaturing polyacrylamide gels, the molecular mass is approximately 38,000 daltons; thus, native AUH is most likely a dimer. A radiolabeled protein extracted from minicells carrying the pKA5 plasmid comigrated with the purified AUH in both sodium dodecyl sulfate-polyacrylamide and native polyacrylamide gels. The pI of purified AUH is between 8.2 and 8.4, as determined by either chromatofocusing or isoelectric focusing. The Km of purified AUH for agmatine is 1.2 mM; the pH optimum is 7.3. Neither the numerous ions and nucleotides tested nor polyamines affected AUH activity in vitro. EDTA and EGTA [ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid] at 1 mM inactivated AUH activity by 53 and 74%, respectively; none of numerous divalent cations tested restored AUH activity. Ornithine inhibited AUH activity noncompetitively (Ki = 6 X 10(-3) M), while arginine inhibited AUH activity competitively (Ki = 9 X 10(-3) M).
Satishchandran
C
C
Boyle
S M
SM
D000376:Agmatine / Q000378:metabolism; D001120:Arginine / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D009711:Nucleotides / Q000494:pharmacology; D009952:Ornithine / Q000494:pharmacology; D011073:Polyamines / Q000494:pharmacology; D011700:Putrescine / Q000096:biosynthesis*; D013696:Temperature; D014512:Ureohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D008670:Metals; D009711:Nucleotides; D011073:Polyamines; D004533:Egtazic Acid; D000376:Agmatine; D001120:Arginine; D004492:Edetic Acid; D009952:Ornithine; D014512:Ureohydrolases; C019625:agmatinase; D011700:Putrescine