Leucine Dehydrogenase is a member of the amino acid dehydrogenase family. Leucine Dehydrogenase is a nicotinamide adenine dinucleotide hydrogen (NADH)-dependent oxidoreductase. It is involved in catalyzing the reductive amination of aliphatic 2-oxo-acids to their respective L-amino acids.
PREPARATION and SPECIFICATION
| Appearance: | White amorphous powder, lyophilized |
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| Activity: | ≥20U/mg enzyme powder (containing approx. 70 % of stabilizers) |
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| Contaminants: | Leucylpeptide decomposing enzymes |
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| (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 |
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PROPERTIES
| Stability | Stable at −20 ℃ for at least one year | (Fig1) |
| Molecular weight | 245,000 |
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| Michaelis constants | 1.0×10⁻³ M (L-Leucine), 3.9×10⁻⁴ M (NAD⁺), 3.5×10⁻⁵ M (NADH), 3.1×10⁻⁴ M [α-Ketoisocaproate (α-KIC)], 2.0×10⁻¹ M (NH₃) |
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| Structure | 6 subunits per enzyme molecule |
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| Inhibitors | Na₂S, Hg²⁺, Cu²⁺, Co²⁺, Mg²⁺, p-chloromercuribenzoate |
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| Optimum pH | 10.5–10.8 (L-Leu→α-KIC), 9.4 (α-KIC→L-Leu) | (Fig3) |
| Optimum temperature | above 70 ℃ | (Fig4) |
| pH Stability | pH 5.5–10.5 (25 ℃, 20 hr) | (Fig5) |
| Thermal stability | below 60 ℃ (pH 6.9, 10 min) | (Fig6) |
| Substrate specificity | (Table 1) |
APPLICATIONS
This enzyme is useful for enzymatic measurement of L-leucine and the activity of leucine amino-peptidase.
ASSAY
1. Principle
The formation of NADH is measured at 340 nm by spectrophotometry.
2. Unit definition
One unit causes the formation of one micromole of NADH per minute under the conditions detailed below.
3. Method
3.1 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
3.2 Procedure
① Prepare the following reaction mixture in a cuvette (d＝1.0cm) and equilibrate at 37 ℃ for approximately 5 minutes.
| 3.0 mL | Substrate solution | (A) |
| 0.3 mL | NAD⁺ solution
| (B) |
Concentration in assay mixture
| Glycine buffer | 0.18 M |
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| L -Leucine | 18 mM |
NAD⁺
| 1.1mM |
② Add 0.05 mL of the enzyme solution* and mix by gentle inversion.
③ Record the increase in optical density at 340 nm against water for 2 to 3 minutes with a spectrophotometer thermostated at 37 ℃, 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) 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) (approx. 5 mg/mL) and dilute to 0.25−0.33 U/mL with the same buffer, immediately before the assay.
3.3 Calculation
Activity can be calculated by using the following formula:
| Vt | : Total volume (3.35 mL) |
| Vs | : Sample volume (0.05 mL) |
| 6.22 | : Millimolar extinction coefficient of NADH (cm²/micromole) |
| 1.0 | : Light path length (cm) |
| df | : Dilution factor |
| C | : Enzyme concentration in dissolution (c mg/mL) |
Table 1. Substrate Specificity of Leucine dehydrogenase
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| Substrate(10mM) | Relative activity(%) | Substrate(10mM) | Relative activity(%) |
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| 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 |
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| 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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