TABLE 2.
Substrate specificity of d-hydantoinase (for hydrolysis reaction) from Blastobacter sp. strain A17p-4a
| Substrate | Km (mM) | Vmax (μmol [min × mg]−1) | Vmax/Km (μmol [min × mg]−1 mM−1) |
|---|---|---|---|
| Cyclic imides | |||
| 2-Methylsuccinimide | 5.7 ± 1.1 | 0.29 ± 0.05 | 0.051 ± 0.004 |
| 2-Phenylsuccinimide | 3.7 ± 0.6 | 0.12 ± 0.02 | 0.032 ± 0.001 |
| Phthalimide | 2.3 ± 0.2 | 0.081 ± 0.01 | 0.035 ± 0.004 |
| 3,4-Pyridine dicarboximide | 11.0 ± 2.0 | 1.2 ± 0.2 | 0.11 ± 0.01 |
| Cyclic ureides | |||
| Hydantoin | 3.2 ± 0.6 | 1.2 ± 0.1 | 0.38 ± 0.04 |
| Dihydrouracil | 2.7 ± 0.1 | 2.5 ± 0.03 | 0.93 ± 0.03 |
| Dihydrothymine | 4.2 ± 1.0 | 1.5 ± 0.4 | 0.36 ± 0.02 |
| dl-5-Methylhydantoin | 9.3 ± 1.9 | 1.6 ± 0.3 | 0.17 ± 0.02 |
| dl-5-(2′-Methylthio-ethyl)hydantoin | 12.0 ± 2.5 | 0.78 ± 0.2 | 0.065 ± 0.009 |
| dl-5-Phenylhydantoin | 6.7 ± 1.2 | 0.84 ± 0.1 | 0.12 ± 0.01 |
| dl-5-(p-Hydroxyphenyl)-hydantoin | 0.61 ± 0.1 | 0.019 ± 0.002 | 0.031 ± 0.005 |
Hydrolysis reactions and analysis were carried out under the standard assay conditions except that each test compound was used as the substrate at varied concentrations (0.1 to 50 mM). No spontaneous hydrolysis of compounds other than phthalimide and 3,4-pyridine dicarboximide was observed. Control reactions without enzyme were performed for these two compounds. The kinetic constant values are averages ± the standard deviation of three different determinations. The following compounds were judged to be inactive as substrates for hydrolysis (less than 10−3 μmol/min/mg of protein with 20 mM each substrate): succinimide, glutarimide, diacetamide, 2,4-thiazolidinedione, rhodanine, 3-methylglutarimide, 3,3-dimethylglutarimide, 3-ethyl-3-methylglutarimide, α,α-dimethyl-β-methylsuccinimide, cycloheximide, N-aminophthalimide, N-methylmaleimide, N-hydrophthalimide, 1,8-naphthalimide, 1,8-naphthalene-dicarboximide, dl-5-carboxy-ethylhydantoin, dl-5-carboxymethylhydantoin, l-carboxymethylhydantoin, l-5-isopropylhydantoin, 1-methylhydantoin, N-methylhydantoin, 5,5-dimethylhydantoin, dihydro-l-orotate, creatinine, barbiturate, dl-pyroglutamate, dl-pyroglutamate methylester, 2-pyrrolidone, allantoin, succinic anhydride, glutaric anhydride, maleic anhydride, ethyleneurea, ethylenethiourea, benzoylurea, propyleneurea, urazole, alloxan, and alloxazine.