4-Guanidinobutyrate amidinohydrolase from Pseudomonas sp. ATCC 14676: Purification to homogeneity and properties

4-Guanidinobutyrate amidinohydrolase (EC 3.5.3.7) was purified about 360-fold to apparent homogeneity from Pseudomonas sp. ATCC 14676. The molecular weight of the enzyme was estimated to be 180,000~186,000 by gel filtration procedure and the method of Hedrick and Smith. The subunit molecular weight was estimated to be 33,000~36,000 by SDS-polyacrylamide gel electrophoresis. The enzyme was optimally active at pH 10.2 in sodium carbonate buffer. EDTA inactivated the enzyme during incubation at 50°C in phosphate buffer (pH 7.0). Incubation of the inactivated enzyme with Mn 2+ restored full activity. The enzyme was inactivated with PCMB, and the PCMB-inactivated enzyme was reactivated by incubation with 2-mercaptoethanol. The enzyme specifically acted toward 4-guanidinobutyrate; 5-guanidinovalerate and 6-guanidinocaproate were hydrolyzed at very low rates. The Km value for 4-guanidinobutyrate was 33 mm. Propionate and n-butyrate were competitive inhibitors of the enzyme with Ki values of 2.0 mm and 0.5 mm, respectively. These properties were compared with those of the analogous guanidinoacetate amidinohydrolase from the same organism.