Bio 1AL- lab 4 (enzymes, seawater platin & vibrio colony streak)

Calculate the initial velocity of β-galactosidase enzyme activity at different concentrations of the substrate, ONPG, and in the presence of an inhibitor

• formula = [cell reference] /0.0023 × 0.0015

• initial rxn velocity is steepest initial slope of ONP curve produced over time

Create Michaelis-Menten and Lineweaver-Burk graphs to communicate the quantitative relationships between ONPG substrate concentration and β-gal activity

michaelis-menton graph and how to find Km/Vmax

reaction rate as a function of substrate concentration (velocity)

• Vmax is where it levels out

• Km is the x value (substrate concentration) corresponding to ½ Vmax

lineweaver-burk graph and how to determine Km/Vmax

• Vmax is reciprocal of y-intercept

• Km is reciprocal of negative X intercept

competitive, noncompetitive, or mixed inhibitor

• competitive- prevent binding of substrate by binding in the substrate parking spot

  • Km INCREASED, Vmax stays same

• noncompetitive- binds reversibly, randomly, and independently of substrate at different sites 

  • Km unaffected, Vmax LOWERS

• allosteric inhibitor (mixed)-

  • Km INCREASES and Vmax LOWERS

β-galactosidase

enzyme whose substrate is lactose

• hydrollyzes lactoseinto glucose and galactose

• must be kept on ice or will be denatured

energy of activation Ea

initial input of energy for an enzyme to convert a substrate

boltzmann distribution

energy distribution of product molecules

• can shift temperature to increase number of molecules with sufficient energy

• can also lower activation energy by using an enzyme

TCEP

buffer to maintain enzyme conformation

• in enzyme solution

• reducing agent to maintain enzyme activity

Michaelis constant Km

rate at which the ES complex dissociates to form either E+S or E+P

• K_m = (k_-1 + k₂) / k₁

• _{low Km = HIGH enzyme substrate affinity}

• _{high Km = LOW enzyme substrate affinity}

IPTG

small molecule which affects B-gal activity

• competitive inhibitor

michaelis menton equation

as substrate concentration increases, the rxn rate approaches Vmax at infinite substrate concentration