Biological Chemistry Laboratory Biology 3515/Chemistry 3515 Spring 2021 Lecture 13:

Burst Substrates and the V vs [S] Experiment

2 March February 2021 ©David P. Goldenberg University of Utah [email protected] The Week Formerly Known as Spring Break

Week of Monday, 8 March

No lab sessions! Lectures on Tuesday and Thursday, as usual. Extra week to prepare lab report for Experiment 3! Use it well! Outline of Enzyme Kinetics Experiment

Last week:

1. Measure velocity as a function of enzyme concentration. 2. Determine enzyme concentration by titration with an inhibitor.

This week:

1. Determine enzyme concentration by reaction with a “burst substrate”. 2. Measure reaction velocity as a function of substrate concentration.

Data analysis. Calculate: • Km • Vmax • kcat Experiment 3, Part C: Another Way to Measure Trypsin Concentration

Based on a historically important experiment:

p-nitrophenyl acetate acetate p-nitrophenol (PNPA)

Reaction with chymotrypsin: PNPA isn’t a very good substrate! Reactions are not linear with time. Initial displacement of curves increases with enzyme concentration. What is going on?

Hartley, B. S. & Kilby, B. A. (1954). The reaction of p-nitrophenyl esters with chymotrypsin and insulin. Biochem. J., 56, 288–297. http://dx.doi.org/10.1042/bj0560288 Proposed Mechanism to Explain “Burst Kinetics”

H3C O + E-Nu O H3C fast slow E-Nu E-Nu O

Enzyme reacts rapidly with substrate to form a covalent intermediate and releases p-nitrophenol. Each molecule of enzyme produces one molecule of product in burst phase. Hydrolysis of the covalent intermediate is much slower and is required to regenerate enzyme (turnover). Steady state rate of product formation is determined by the second step. Among the first evidence for a covalent intermediate in an enzyme-catalyzed reaction. A Designed Burst Substrate for Trypsin

+

Enzyme Enzyme

Guanido group resembles arginine side chain. Hydrolysis of covalent intermediate is very slow. (half-time greater than ≈ 40 h) An (almost) irreversible inhibitor, or “suicide substrate”. Why is hydrolysis so slow? Resonance Stabilization of the Covalent Intermediate

NH2 NH2

H2N O H2N O HN N

O O

Enzyme Enzyme

Resonance structures shift electrons from phenyl ring to carbonyl carbon. Electron density on carbonyl carbon disfavors nucleophilic attack by water. Why doesn’t the same effect prevent formation of the intermediate? Resonance Also Favors Formation of the Covalent Intermediate

NH2

H2N O HN +

O NO2

Enzyme Enzyme

Resonance shifts electrons from carbonyl carbon to nitrophenol ring. Makes the nitrophenol group a better leaving group. Favors nucleophilic attack in the first step of the reaction. Three-dimensional Structure of p-guanido benzoate intermediate

As predicted, Ser 195 O‚ forms ester with p-guanido benzoate. Gly 193 Amide groups of Ser 195 and Gly 193 form “oxyanion hole” that stabilizes negative charge Ser 195 on carbonyl oxygen in transition state. His 57 Where would the water molecule be for hydrolysis of the intermediate? Asp 102

Mangel, W. F., Singer, P. T., Umland, T. C., Toledo, D. L., Stroud, R. M., Pflugrath, J. W. & Sweet, R. M. (1990). Structure of an acyl-enzyme intermediate during catalysis:(guanidobenzoyl)trypsin. Biochemistry, 29, 8351–8357. http://dx.doi.org/10.1021/bi00488a022 Experiment 3, Part C: Measurement of Trypsin Concentration with Burst Substrate

0.2 "Burst"

Spontaneous hydrolysis

402 0.1 A

0 0 1 2 3 4 5 Time (min)

Requires high enzyme concentration, since each enzyme molecule generates only one chromophore molecule. Tris buffer is bad for this experiment, because amines are nucleophilic and react with p-NPGB. Need to record both the absolute increase in absorbance and the rate of steady-state increase, in order to extrapolate initial burst phase. May need to ignore the first data point, since it is recorded slightly after reaction starts, but possibly before the burst is complete. Experiment 3, Part D: Velocity as a Function of Substrate Concentration

To reliably estimate both Km and Vmax, substrate concentrations must cover range both below and above Km. We will use eleven substrate concentrations, plus a control without substrate. Velocity (M/min) Velocity Two groups of six reactions. [S] (M) How should the reactions be grouped? Two Ways to Group the Reactions

50 50 Group 1 Group 1 Group 2 Group 2 40 40

30 30 V V

20 20

10 10

0 0 0 100 200 300 400 0 100 200 300 400 [S] (µM) [S] (µM)

Is one way better than the other? What if something changes between the reactions? Two Ways to Group the Reactions

50 50 Group 1 Group 1 Group 2 Group 2 40 40

30 30 V V

20 20

10 10

0 0 0 100 200 300 400 0 100 200 300 400 [S] (µM) [S] (µM)

1–6, 7–2 grouping: changes between reaction groups may be hard to detect. Odd–even grouping: changes between reaction groups are easier to detect. Fitting data together averages effects more evenly. Dilutions of Trypsin Solutions for Experiment 3 Warning!

Direction Change Data Analysis for the Michaelis-Menten Experiment Analysis of Data from the V versus [S] Experiment Velocity

Substrate Concentration

We want to fit the experimental data to the Michaelis-Menten Equation:

[S]V V = max [S] + Km

From the fit, we obtain estimates of Km and Vmax. Clicker Question #1

Estimate Vmax from the graph:

40 A) 30 nM/s

B) 40 nM/s ∗

20 C) 50 nM/s D) 80 nM/s ∗close enough for credit.

0 0 2 4 6 Clicker Question #2

Estimate Km from the graph:

40 A) 1 —M

B) 2 —M

C) 5 —M 20 D) 10 —M

0 0 2 4 6