LEUCINE DEHYDROGENASE from Bacillus sp. ¹⁾

LED-201

PREPARATION and SPECIFICATION
Appearance:	White amorphous powder, lyophilized
Activity:	GradeⅡ 20U/mg-solid or more (containing approx. 70% of stabilizers)
Contaminants:	Leucylpeptide decomposing enzymes (Leu-Val) ≤1.0×10⁻²% (Leu-Gly-Gly) ≤1.0×10⁻²% NADH oxidase ≤1.0×10⁻²%
Stabilizers:	2-Mercaptoethanol, L-cysteine, dithiothreitol, ethylenediaminetetraacetate

PROPERTIES
Stability:	Stable at -20°C for at least One year (Fig.1)
Molecular weight ² ⁾:	245,000
Michaelis constants ² ⁾:	1.0×10⁻³M (L-Leucine), 3.9×10⁻⁴M (NAD⁺), 3.5×10⁻⁵M (NADH), 3.1×10⁻⁴M [α-Ketoisocaproate (α-KIC)], 2.0×10⁻¹M (NH₃)
Structure ² ⁾:	6 subunits per enzyme molecule
Inhibitors ² ⁾:	Na₂S, Hg⁺⁺, Cu⁺⁺, Co⁺⁺, Mg⁺⁺, p-chloromercuribenzoate
Optimum pH :	10.5-10.8 (L-Leu→α-KIC), 9.4 (α-KIC→L-Leu )(Fig.3)
Optimum temperature :	above 70°C(Fig.4)
pH Stability :	pH 5.5-10.5 (25°C, 20hr)(Fig.5)
Thermal stability :	below 60°C (pH 6.9, 10min)(Fig.6)
Substrate specificity:	(Table 1)

APPLICATIONS ⁴⁾

This enzyme is useful for enzyme determination of L-leucine and the activity of leucine amino- peptidase.

ASSAY

Principle:

leucine dehydrogenase

L-Leucine+NAD⁺+H₂O ►α-Ketoisocaproate+NADH+NH₃+H⁺

The appearance of NADH is measured at 340nm by spectrophotometry.

Unit definition:

One unit causes the formation of one micromole of NADH per minute under the conditions described below.

Method:

Reagents
A. L-Leucine solution:	20mM L-leucine in 0.2M glycine-KCl-KOH buffer, pH 10.5 (Prepare freshly)
B. NAD⁺ solution:	12.5mM (Should be prepared fresh)
C. Enzyme diluent:	25mM K-phosphate buffer, pH 7.2

Procedure

Concentration in assay mixture
Glycine buffer	0.18 M
L-Leucine	18m M
NAD⁺	1.1mM

1. Prepare the following reaction mixture in a cuvette (d=1.0cm) and equilibrate at 37°C for about 5 minutes.

3.0ml Substrate solution (A)
0.3ml NAD⁺ solution (B)

2. Add 0.05ml of the enzyme solution* and mix by gentle inversion.

3. Record the increase in optical density at 340nm against water for 2 to 3 minutes in a spectrophotometer thermostated at 37°C, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔOD test).
At the same time, measure the blank rate (ΔOD blank) by using the same method as the test except that the enzyme diluent (C) is added instead of the enzyme solution.

* Dissolve the enzyme preparation in ice-cold enzyme diluent (C) (ca. 5mg/ml) and dilute to 0.25−0.33U/ml with the same buffer, immediately before assay.

Calculation

Activity can be calculated by using the following formula :

ΔOD/min (ΔOD test-ΔOD blank)×Vt×df

Volume activity (U/ml) = = ΔOD/min×10.77×df

6.22×1.0×Vs

Weight activity (U/mg)=(U/ml)×1/C

Vt: : Total volume (3.35ml)
Vs: : Sample volume (0.05ml)
6.22: : Millimolar extinction coefficient of NADH (㎠/micromole)
1.0: : Light path length (cm)
df: : Dilution factor
C: : Enzyme concentration in dissolution (c mg/ml)

REFERENCES

K.Soda et al.; Biochem.Biophys.Res.Commun., 44, 931 (1971).
T.Ohshima et al.; J.Biol.Chem., 253, 5719 (1978).

**Table 1. Substrate Specificity of Leucine dehydrogenase ² ⁾**
Substrate (10mM)	Relative activity(%)	Substrate (10mM)	Relative activity(%)
L-Leucine	100	α-Ketoisocaproate	100
L-Valine	74	α-Ketoisovalerate	126
L-Isoleucine	58	α-Ketovalerate	76
L-Norvaline	41	α-Ketobutyrate	57
L-Norleucine	10	α-Ketocaproate	46
L-Methionine	0.6	Inert: Pyruvate, α-Ketoglutarate, Phenylpyruvate, Oxaloacetate, Glyoxylate
L-Cysteine	0.3
Inert: L-Ala, L-Glu, L-Thr, L-Ser Gly, L-Phe, L-Lys, L-Arg, D-Leu, D-Val, D-Ile

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