TABLE 3.

Substrate specificity of half-amidase from Blastobacter sp. strain A17p-4^a

Substrate	Km (mM)	kcat (s−1)	kcat/Km (s−1 mM−1)
Succinamic acid	6.2 ± 0.4	5.76 ± 0.30	0.93 ± 0.02
Glutaramic acid	2.8 ± 0.1	2.23 ± 0.10	1.62 ± 0.06
Adipiamic acid	8.0 ± 1.4	0.36 ± 0.05	0.046 ± 0.002
Lactamide	3.7 ± 0.3	0.11 ± 0.01	0.033 ± 0.002
n-Valeramide	5.1 ± 0.2	0.18 ± 0.01	0.037 ± 0.006
n-Caproamide	17.0 ± 2.7	0.18 ± 0.04	0.011 ± 0.001
Crotoamide	8.8 ± 0.4	0.10 ± 0.01	0.012 ± 0.001
Benzamide	6.5 ± 0.1	0.13 ± 0.01	0.020 ± 0.001
2-Phenylpropioamide	3.1 ± 0.1	0.078 ± 0.002	0.026 ± 0.001

^aReactions were carried out under standard assay conditions described in Materials and Methods with the variation of substrates, and the amount of ammonia produced was colorimetrically estimated by indophenol method. As for the ester substrates, the reduction of ester or the production of acid was determined by HPLC as described in Materials and Methods. The kinetic constant values are averages ± standard deviations of three separate determinations. The following compounds were judged to be inactive as substrate (less than 0.1% k_cat values for succinamic acid; <0.005 s⁻¹): acetamide, fluoroacetamide, chloroacetamide, propioamide, n-butyramide, isobutyramide, acrylamide, oleamide, stearamide, nicotinamide, 2-thiophenecarboxamide, o-aminobenzamide, o-chlorobenzamide, 4,5-imidazoledicarboxamide, o-toluamide, phthalamidic acid, salicylicamide, dl-phenylalanine amide, glycine amide, glycyl-dl-alanine, asparagine, glutamine, fumaramide, succinamide, adipiamide, α-ketoglutaramic acid, α-ketosuccinamic acid, urea, ethylurea, N-methylurea, oxamide, biurea, hydantoic acid, β-ureidopropionate, N-carbamoyl-d- and -l-amino acids, allantoin, pyrazine carboximide, 4-pyridine carboxylate amide, succinate monomethyl ester, succinate monoethyl ester, lactic acid ethyl ester, crotonic acid methyl ester, and isovaleric acid ethyl ester.