lecture 7 - enzyme kinetics

what is enzyme kinetics

study of how enzymes bind substrates and turn them into products

is the step E + S → ES reversible or irreversible

reversible

michaelis menten equation

• V = velocity of reaction

• V_max = maximum rate achieved by the system

• [S] = concentration of a substrate S

• K_M = michaelis constant

michaelis constant (Km)

Km has the same units as substrate concentration

what does a low Km mean

the enzyme has high affinity for substrate

• active even at low substrate levels

what does a high Km mean

enzyme needs more substrate to reach half maximal activity

michaelis menten vs Vmax

• low [S] - enzyme activity increases sharply with more substrate

• very high [S] - rate approaches a maximum (Vmax) and levels off

• low Vmax enzyme - overall reaction is slow

what is Vmax

the fastest rate the enzyme can achieve at saturating substrate

lineweaver burk equation

lineweaver burk plot

• double reciprocal plot of 1/V (rate) vs 1/[S] (substrate)

• turns curved MM plot into straight line

• Y-intercept (ordinate) = 1/Vmax

  • helps to find max speed of enzyme

• X-intercept (abscissa) = -1/Km

  • helps find the enzyme’s affinity for substrate

what is the main advantage of lineweaver burk plot

makes it easier to calculate Vmax and Km

turnover number (Kcat)

• Kcat = Vmax / [Et]

  • Et = total enzyme concentration

• tells how many product molecules are formed per enzyme active site per second

• stays constant even as an enzyme is purified

specific activity

enzyme activity per mg of protein

• increases as enzyme is purified (less contaminating protein)

competitive inhibition

competes with substrate for binding to the active site

• Km increases, but Vmax stays the same

• changes Km only

non competitive inhibition

binds to a different site, not the active site

• Vmax decreases, but Km does not change

• changes Vmax only

what does Km measure

measures substrate concentration needed for half maximal activity

what does a low Km mean

• high affinity

• enzyme effective at low substrate levels

what is Vmax

maximum rate at saturating substrate

specific activity does what with purification

increases

in addition to the basic ES intermediate in an enzyme reaction, what other types of intermediates are formed and why are they important