Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species. A hydantoinase with no 5,6-dihydropyrimidine amidohydrolase activity.
A soluble hydantoinase (5,6-dihydropyrimidine amidohydrolase) was purified to homogeneity from a newly isolated Agrobacterium species. This hydrolase consists of about 578 aminoacyl residues and is a slightly acidic protein with an isoelectric point of 6.5. The first 22 N-terminal amino acid residues were determined by Edman degradation. Determination of the relative molecular mass of the protein by gel-filtration chromatography gave an apparent value of 250,000. The subunit M(r) was 62,000, as estimated by analytical SDS/PAGE and 66,500, as estimated by denaturing gel-filtration chromatography. The pure hydantoinase exhibits the following hydrodynamic properties: a sedimentation coefficient of 8.8 S as determined by sedimentation velocity experiments; a Stokes radius of 6.8 nm; a diffusion coefficient of 31.5 microns2.s-1 as determined by analytical gel-filtration chromatography. From these experimental data, the following physical constants could be calculated: a theoretical M(r) of 265,000, a frictional ratio, f/fo, of 1.59, a maximal axial ratio, a/b, of 3.1; a Perrin shape factor, F, of 1.37. As shown by different Km values, the preferred substrates of this hydrolase were 5-monosubstituted hydantoins bearing aromatic substituents. 5,5-Dimethylhydantoin and different thio analogs of the 5-p-hydroxyphenylhydantoin molecule are competitive inhibitors of this hydrolase. The classification of this microbial hydantoinase, which exhibits no hydrolytic activity with all the dihydropyrimidines tested, under the systematic name of 5,6-dihydropyrimidine amidohydrolase, and its putative metabolic role are further discussed.
Runser
S M
SM
Institut de Chimie des Substances Naturelles, Centre National de la Recherche Scientifique, Gif-Sur-Yvette, France.
Meyer
P C
PC
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D004058:Diffusion; D006827:Hydantoins / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D012231:Rhizobium / Q000201:enzymology*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D006827:Hydantoins; D000581:Amidohydrolases; C020047:dihydropyrimidinase