PURIFICATION AND BIOCHEMICAL-CHARACTERIZATION OF THE HYDANTOIN HYDROLYZING ENZYME FROM AGROBACTERIUM SPECIES - A HYDANTOINASE WITH NO 5,6-DIHYDROPYRIMIDINE AMIDOHYDROLASE ACTIVITY

被引:68
作者
RUNSER, SM [1 ]
MEYER, PC [1 ]
机构
[1] CNRS,INST CHIM SUBST NAT,F-91190 GIF SUR YVETTE,FRANCE
来源
EUROPEAN JOURNAL OF BIOCHEMISTRY | 1993年 / 213卷 / 03期
关键词
D O I
10.1111/j.1432-1033.1993.tb17883.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 250000. The subunit M(r) was 62000, as estimated by analytical SDS/PAGE and 66500, 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 mum2.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 265000, a frictional ratio, f/fo, of 1.59, a maximal axial ratio, alb, of 3.1; a Perrin shape factor, F, of 1.37. As shown by different K(m) 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.
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页码:1315 / 1324
页数:10
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