Enzyme Kinetics of Salivary α-Amylase: Determination of Km and Vmax
Salivary $alpha$-amylase ($alpha$-1,4-glucan 4-glucanohydrolase, EC 3.2.1.1) is a calcium-dependent metalloenzyme that endo-cleaves internal $alpha(1to 4)$ glycosidic linkages in amylose and amylopectin, releasing maltose, maltotriose, and $alpha$-limit dextrins. The rate of maltose formation follows the Michaelis-Menten equation:
Where $v_0$ is initial velocity, $V_{max}$ is maximal velocity at enzyme saturation, and $K_m$ (Michaelis constant) represents the substrate concentration at which velocity is half-maximal ($v_0 = V_{max}/2$). Taking the reciprocal yields the linear Lineweaver-Burk double reciprocal equation:
Maltose generated by enzymatic hydrolysis is quantified using the 3,5-dinitrosalicylic acid (DNSA) assay. Under alkaline boiling conditions, maltose reduces DNSA (yellow) to 3-amino-5-nitrosalicylic acid (ANSA, dark reddish-brown), measured spectrophotometrically at $lambda = 540,text{nm}$.
| Tube No. | Starch Conc. [S] (mg/mL) | 1 / [S] (mL/mg) | Measured A540 | Maltose Produced (μmol) | v0 (μmol/min) | 1 / v0 (min/μmol) |
|---|---|---|---|---|---|---|
| 1 | 0.20 | 5.00 | 0.145 | 0.29 | 0.029 | 34.48 |
| 2 | 0.40 | 2.50 | 0.250 | 0.50 | 0.050 | 20.00 |
| 3 | 0.80 | 1.25 | 0.390 | 0.78 | 0.078 | 12.82 |
| 4 | 1.60 | 0.625 | 0.540 | 1.08 | 0.108 | 9.26 |
| 5 | 3.20 | 0.312 | 0.670 | 1.34 | 0.134 | 7.46 |
| 6 | 6.40 | 0.156 | 0.760 | 1.52 | 0.152 | 6.58 |
Plotting $1/v_0$ vs $1/[S]$ yields linear slope $m = K_m/V_{max} = 5.76$ and y-intercept $c = 1/V_{max} = 5.68,text{min}/mutext{mol}$:
$$V_{max} = frac{1}{c} = frac{1}{5.68} = 0.176,mutext{mol/min}$$
$$K_m = text{Slope} times V_{max} = 5.76 times 0.176 = 1.01,text{mg/mL}$$
