Enzyme Kinetics

* Experimental Objective(s) – The objectives of this lab experiment are to determine enzymatic kinetic parameters for the reduction of pyruvate by LDH such as reaction rates based on experimental time course data, and plotting standard curves of substrate, Michaelis-Menten & Lineweaver-Burk plots also using experimental data. Furthermore, enzyme activity at varying substrate concentrations along with the effect of an inhibitor and the type of inhibition will be studied.

* Results:

(A) Initial Rate Data

| Unit | Value |

Rate | ABS/min | -0.0072 |

r2 | - | 0.996 |

Wavelength | nm | 340 |

To convert ABS/min to M/min (or mM/min), Beer Lambert’s law will be used.

A = εlc

∴ c=Aεl

c= -0.0072 min-16220 M-1cm-1 ×1 cm

= -1.16×10-6Mminor-0.0016 mM/min

(B) Sketching Standard Curve for Substrate Data

Substrate Concentration (mM) | Absorbance @ 510 nm (ABS) |

0.01 | 0.026 |

0.025 | 0.077 |

0.05 | 0.164 |

0.1 | 0.299 |

0.2 | 0.548 |

0.4 | 1.241 |

0.6 | 1.756 |

(C) Relationship between reaction rate and substrate concentration

1 - Sketching a Plot of Reaction Rate Versus Concentration of Pyruvate

Substrate Concentration (mM) | Absorbance @ 510 nm (ABS) |

0.05 | 0.125 |

0.1 | 0.254 |

0.2 | 0.529 |

0.4 | 1.124 |

0.6 | 1.712 |

| Residual concentration of pyruvate in each tube & Inverse of v and [S] Linear Equation: y = 2.963x + 0.001 [x=concentration; y=absorbance] |

Original Conc. (mM) - x | 0.05 | 0.1 | 0.2 | 0.4 | 0.6 |

Absorbance - y | 0.125 | 0.254 | 0.529 | 1.124 | 1.712 |

Residual Conc. (mM) - r: x= y-0.0012.963 | 0.125-0.0012.963= 0.042 | 0.254-0.0012.963= 0.085 | 0.529-0.0012.963= 0.18 | 1.124-0.0012.963= 0.379 | 1.712-0.0012.963= 0.58 |

Reaction rate (mM/min) - vv= x-r5 mins | 0.05-0.0425= 0.0016 | 0.1-0.0855= 0.003 | 0.2-0.185 = 0.004 | 0.4-0.3795 = 0.0042 | 0.6-0.585 = 0.0045 |

1/v | 1/0.0016= 625 | 1/0.003= 333.3 | 1/0.004= 250 | 1/0.0042= 238.1 | 1/0.002= 222.2 |...