Previous Class. Reasons for analyzing pre-steady state conditions Methods for pre-steady state measurements. Today

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1 Previous Class Reasons for analyzing pre-steady state conditions Methods for pre-steady state measurements Today Spectrophotometry Spectrofluorimetry Radioactive Procedures ph dependency

2 Spectrophotometry A spectrophotometer is employed to measure the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector A = log I0/I What are the factors that affect the amount of light a sample absorbs? Beer's Law (1852) : The absorbance displays a simple dependence on the concentration of the analyte and the cell path length A = εcl Where ε is the molar absorptivity, c is the concentration, and l is the length of sample that the light passes through (in cm)

3 Is a measure of the ability of an analyte to absorb light at a specified wavelength. (find λ max) Is supposed to be constant for Beer s law to be valid DETERMINE ε Molar Absorptivity Determination Measure the intensity of absorbed light for solutions with various concentrations of analyte. Construct a plot of A vs c (should be a straight line) Plot the line-of-best-fit through the experimental points. The slope of this line is the molar absorptivity (at a particular path length)

4 Molar Absorptivity Determination ε = L/mol cm

5 Spectrofluorimetry Fluorescent compounds absorb light (photon) to give excited state then re-emits light (photon) during decay at a different (usually longer) wavelength Fluorimetry is usually much more sensitive than spectrophotometry Concentration of product is obtained using standard curve

6 Radioactive Procedures Most sensitive assay methods involve radioisotopes (orders of magnitude more sensitive than spectrophotometry) Common isotopes include P 32, C 14, H 3, S 35 ATP all phosphate groups can be labeled (gamma most common) Radioactive decay (measured in cpm) involves the emitting of electrons that are converted to light quanta by a scintillant solution P 32 can spontaneously emit photons and can be captured by film detector Often radioactive methods are used to examine enzymes in vivo to provide qualitative data (but can be quantitative also)

7 Kinase Enzyme Assay from Cells Lyse Transfected cells and remove cell debris Immunoprecipitate protein kinase of interest Incubate with γ-[ 32 P] ATP and substrate SDS-PAGE Autoradiography

8 Enzyme activity of MLK3 kinase and a MLK3 oligomerization mutant MLK3: - WT L410P MLK3 P histone P substrate

9 Mitogen Activated Protein Kinase Signaling Cascades

10 Kinase Assay Lyse Transfected cells and remove cell debris Immunoprecipitate protein kinase of interest Incubate with γ-[ 32 P] ATP and substrate SDS-PAGE Autoradiography

11 Enzyme activity of Downstream Kinase (JNK) MLK3: - WT L410P JNK IP substrate c-jun P Most Sensitive!!! Experiment from one plate of human cells [E] = fmol vs Spectrophotometry [E] = µmol

12 Next Topic ph dependence of Enzyme catalysis

13 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

14 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

15 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 + ]

16 ph Effects on Enzyme Activity

17 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

18 Analysis of Single Ionizing Group Km kcat Ka E EH+S K m Ka ES EHS Assumptions: 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

19 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

20 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

21 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

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

23 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

24 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)

25 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

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

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

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

29 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

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