Previous Class. Today. Spectrophotometry Spectrofluorimetry Radioactive procedures. ph dependence of Enzyme Catalysis (focus on pgs.

Transcription

1 Spectrophotometry Spectrofluorimetry Radioactive procedures Previous Class Today ph dependence of Enzyme Catalysis (focus on pgs )

2 ph Effects on Enzyme Activity Structural Considerations: Extreme ph changes will denature folded structure due to repulsive forces Milder ph changes can dissociate oligomeric state of enzyme into inactive monomers Therefore, important to be aware of protein stability issues due to ph conditions when characterizing enzyme activity

3 ph Effects on Enzyme Activity Catalytic Considerations: Substrate Ionization may affect enzyme-substrate productive binding Enzyme catalytic groups contain acid and basic groups; therefore, ionization state critical for proper catalysis Km productive substrate binding : ion pair, hydrogen bonding, hydrophobic interactions kcat Chemical transformation : Acid/Base, Nucleophilic catalysis Electrostatic catalysis

4 Acid-Base Chemistry HA + B A - + BH + The maximum activity at a given [E T ] and [S] depends on the ratio of [A]/[HA] or [B]/[BH]. Therefore, the effects of ph on enzyme activity can be expressed in terms of the Henderson-Hasselbach equation ph = pka + log[a - ]/[HA] ph = pka + log[b]/[bh + ]

5 ph Effects on Enzyme Activity

6 How pka s are Perturbed A)Ionizable groups are buried in hydrophobic environment: Causes groups to take on neutral form The pka of acidic groups increases The pka of basic groups decreases B) Ionizable groups are exposed to electrostatic environment For opposite charge environment pka of acidic groups decrease pka of basic groups increase Like charge environ. increase decrease

7 Analysis of Single Ionizing Group Km kcat Ka E EH+S K m Ka ES EHS Assumptions: Groups act as titrating acids and bases Only one ionization state of the enzyme is active (ES) The enzyme is stable over the ph range studied The proton transfer steps are much faster than the chemical steps The rate determining step doesn't change with ph

8 Analysis of Single Ionizing Group Suppose one group on the enzyme has to be unprotonated for activity: Titrating ionizing group into form essential for activity Midpoint represents the pka of ionizing group v 0 [E] [EH] pka ph

9 Analysis of Single Ionizing Group Similarily, suppose one group on the enzyme has to be protonated for activity: Titrating ionizing group into inactive form Midpoint represents the pka of ionizing group v 0 [EH] [E] pka ph

10 Analysis of Two Ionizing Groups Frequently there are two groups that need to be in a correct ionizable form for proper catalytic activity For example: a protonated Asp (catalytic acid), and a unprotonated Cys (catalytic nucleophile) Velocity versus ph plot results in a bell shaped curve

11 Analysis of Two Ionizing Groups ph Optimum v 0 Half maximum for given [S] and [E] pka pka ph

12 The dependency of ph How the enzyme activity is altered by ph dependency may allow identification of the type of groups present at the active site that are important for catalysis (aware of stability issues) Structure could change due to ionic interactions Catalytic groups not in proper protonated form to carry out chemical transformation steps

13 The dependency of ph Simplified insights: 1) If the ph dependent process affects the transformation of: ES E + P Then Vmax and kcat will be affected, dependent on the pka of the ES complex 2) If the ph dependent process affects the transformation of: E+S ES Then the Km of the reaction will be affected, dependent on the pka of the E, S, (forward) and ES complex (reverse)

14 The dependency of ph Simplified insights: 3) If the ph dependent process affects the transformation of: E+S E + P Then the kcat/km of the reaction will be affected, dependent on the pka of the E, and S

15 The dependency of ph on Enzyme Catalysis Why is chymotrypsin most active at ph 8? Catalytic Triad mechanism pka ~4 pka ~6.8

16 The dependency of ph on Enzyme Catalysis Why is chymotrypsin most active at ph 8? Expect activity to be maximum above ph 7

17 The dependency of ph on Enzyme Catalysis Why is chymotrypsin most active at ph 8? Secondary effect

18 The dependency of ph on Enzyme Catalysis Why is chymotrypsin most active at ph 8? Secondary effect A properly protonated NH 3+ terminal Ile 16 is necessary to position Asp 192 for a proper substrate binding backbone (ion pair contribution) The pka of the α NH 3+ group is ~9-10 Therefore expect the activity to decrease at ph conditions above ph 9 due to an increase in Km

19 The dependency of ph on Enzyme Catalysis Why is chymotrypsin most active at ph 8? Secondary effect