Lecture 11: Enzyme Kinetics: km,Kcat,Km/Kcat,Ki, + Competitive Inhibtior

Malonate

Compeitive Inhibitor of SDH: Competes with Succinate at the active site of Succinate Dehydrogenase and won't be proccessed by the enzyme

CI stands for

Competitive Inhibitor

Benazmidine

Competitive Inhibitor of Trypsin. (in handout pg.9 you'll notice similarity between Benzamidine and Arginine, the latter of course is a substrate of Trypsin. Hence, they both will compete for the active site of tryspin.

State the Equation for Competitive Inhibitors

E.I double arrow (equilbiirum) giving us E + I E.I. is the enzyme inhibitor complex; double arrow in the different directions because it is reversible, and then the products are the Enzyme and the Inhibitor.

Ki is a type of

Equilibrium Constant or Keq (hence it will be equal to products over reactants)

Ambiguous but possible test question - We can use Km to compare the affinities of different substrates to different enzymes. (T or F)

False - We can use Km to compare the affinities of different substrates TO THE SAME ENZYME (you're comparing their binding strengths)

Km = 1/2 Vmax (T or F)

False. It is the Substrate CONCENTRATION when you are at ½ Vmax.

Irreversible Inhibitor: (what's the keyword)

Involves COVALENT BONDING between the inhibitor and the enzyme.

Define Km

It is the Substrate CONCENTRATION when you are at ½ Vmax.

Why is specificity constant more accurate than Km in telling us about the catalytic efficinecy of the enzyme. What are the units?

It takes into account: i. Binding (enzyme has to get together with the substrate) - which is kM ii.Turnover (conversion of the complex to the product) which is Kcat. Units: #/[M*s] (can be derived bec. Kcat = #/seconds)

Catalytic efficiency of the enzyme is measured by; turnover number is measured by, how tightly the enzyme binds a specfic substrate.

Kcat/Km (specificty constant); kcat, km

Calcualting Ki

Ki is a Keq and hence it is = Products over reactants Therefore Keq or Ki = E + I / E.I. Higer E.I means lower Ki which explains why a low Ki indicates a potent inhibitor that has high affinity for the enzyme.

Apparent Km. Why is it called so?

Km is indicative of the affinity of the enzyme to the substrate. In the case of adding CI, the affinity hasn't changed at all. When we say that Competitive Inhibitors increase Km, we say that it increased the apparent Km because the affinity hasn't changed at all but rather that we need to increase the substrate concentration so that we can reach 1/2 Vmax.

Difference between Km and Kcat/Km

Km tells you how tightly the enzyme binds to the substrate but this is not enough to allow you to predict how fast or readily the product will form. Kcat/Km on the other hand takes into account the binding affinity of the substrate to the enzyme (Km) IN ADDITION to how readily the substrate-enzyme complex can form the product.

LBP stands for; what is the function?

Lineweaver-Burk Plot. (not lukewaver) We can use this plot to distinguish between the 2 types of reversible inhibitors: Competetive and Non-competetive Inhibitors.

Hint: how would you recognize Km

Looking at the value or parameter that is in M, μM or mM.

Why do we care about inhibitors? In other words Why are we studying them.?

Many drugs are inhibitors.

Theoretically, why doesn't the CI alter Vmax.

No because Vmax represents the maximums speed the enzyme is working at. This speed will increase with increasing Km or increasing substrate concentrations at the enzyme binding site so more substrates can be converted to products per unit time. However at a certain Km or [S], all the enzyme binding sites are full and hence it its Vmax will plateau at the point forward. We can always counteract the addition of CI by increasing Substrate concentration and hence the enzyme can still work at their maximum speed even in the presence of CI.

Non-competetive Inhibitor is a kind of

REVERSIBLE inhibitor.

Vmax (define, what is the substrate concentration here?)

Rate of the enzyme-catalyzed reaction when the enzyme is fully saturated. Explanation: Vmax represents the maximums speed the enzyme is working at. This speed will increase with increasing Km or increasing substrate concentrations at the enzyme binding site so more substrates can be converted to products per unit time. However at a certain Km or [S], all the enzyme binding sites are full and hence it its Vmax will plateau at the point forward.

ii. 2 types of inhibitors.

Reversal and Irreversible Inhibitors.

If the enzyme inhibitor kills the person and is capable of dissociating from the enzyme, the inhibitor is (irreversible, reversible)

Reversible. As long as it can still come off from the enzyme even if person dies it is reversible inhihbitor = NOT irreversible.

Units of Km

Since it is a substrate concentration: it will have units of microMolar, milliMolar or Molar.

Your body can increase or decrease the substrate concentration. This can affect the ......... of the enzyme.

Speed, that's why we say that the enzyme is operating at its Vmax when its fully saturated.

High Km means:

Substrate binds loosely to the enzyme.

Converting E to ES complex means; state the equation

That we will irreverisbly be able to produce the product from the substate. E + S double equilibrium arrow E.S (E.S. Complex) IRREVERSIBLE arrow towards: E + P.

What is a wild type enzyme

The "normal" enzyme. The enzyme that prevails among individuals in natural conditions, contrary to the a atypical mutant type.

HMG-COa Reductase: the rate limiting step of cholesterol.

The enzyme that carries the rate-limiting step of Cholesterol synthesis. Need to remember this enzyme because this is the enzyme that drugs will work on to shut down cholesterol synthesis.

Mnemonic to remember where hexokinase is.

The fact that it is called hexokinase means it is not specific enough which also corresponds to the low affinity (not enough specificity) it has to the sugar substrate. Hence this will be the one in the liver because the cells will need the other enzyme that has high affinity to glucose especially when it really needs a rapid supply of energy fuel.

Ki (not Km)

The inhibition constant of the COMPETITIVE inhibitor.

Reversal inhibitors (talk about the bonds they make with the enzyme)

They make all sort of non-covalent bonds with the enzyme.

A competitive inhibitor increases Km. (T or F). This is because it decreases the binding affinity of the enzyme to the substrate. (T or F)

True; False. Although Km is indicative of the affinity of the enzyme to the substrate, the affinity hasn't changed at all. When we say that Competitive Inhibitors increase Km, we mean we are increasing the substrate concentration needed so that we can reach 1/2 Vmax.

Equation to calculate Kcat - We won't be asked to do problems that ask us to calculate Kcat but recognize how it will look like

Vmax/ ET Vmax over the total Enzyme Concnetration or ET. OR (# of moles of product converted from substrate per mole of enzyme-substrate complex) per unit time Example ........ per unit time like min-1 or day-1.

When can we use Km value as a measure of affinity

We can use Km to compare the affinities of different substrates TO THE SAME ENZYME (you're comparing their binding strengths)

Units for Kcat, Comment on Kcat for acetylcholinesterase, how to recognize it in on the exam.

[ #/ seconds, days, etc.], 3000 sec^-1, that's how it will be shown

What's the equation in LWB Plot? y = mx + b

[1/vo] = [Km/Vmax] [ (1/S) ] + [1/Vmax]

2 types of Reversible Interactions with their abbreviations.

a. Competitive Inhibitor (CI) b. Non-Competitive Inhibitor (NCI)

Hexokinases are present in ............ (mnemonic) vs Glucokinase present in

all cells except the liver (mnemonic - notice how it is a general term) , liver only

- 5FU is (what kind of inhibitor, the enzyme that is inhibitred)

an irreversible inhibitor of Thymidylate synthase. The only enzyme that allows cancer cells to synthetize Thymadine which is important for DNA synthesis and replication and hence cell survival.

Enzyme Inhibition: An inhibitor:

any compound that binds to an enzyme and interferes with its activity by either slowing down the activity of the enzyme (Like NCI) or completely stopping it.

Define Isoenzymes, give examples

are enzymes that catalyze the same chemical reaction but have different amino acid sequences (and hence different structures) Hexokinases in all cells except the liver and Glukokinases in only the liver cells.

Trypsin

cleaves at the carboxy terminal of arginine and lysine (except when either are followed by proline) from wikipedia but good to know.

Chemotrypsin (cleaves where)

cleaves at the carboxy terminal of tyrosine, tryptophan, and phenylalanine

Indole; how do we recognize it is a competitive inhibitor on the exam (important - WILL BE ON THE EXAM)

competitive inhibitor of chymotrypsin enzyme; *** In the handout pg.9 you'll notice that satisfies all conditions of a competitive inhibitor. a) Indole looks like tryptphan in terms of the aromatic rings. b) Will competete with tyryptophan at the active site of chemotrypsins. c) It won't be processed by the enzyme chemotrypsin and won't do anything except blocking the active site preventing tryptophan from binding.

Both 5-Fluorouracil and Methotrexate are

enzyme inhibitors. Together: MX and 5FU will shut down DNA synthesisi and then you kill cells because they can't divide anymore.

1. Under some conditions, Km can be also a measure of

how tightly or loosely the substrate binds to the enzyme.

The substrate that the enzyme processes most efficiently is the one with the highest

kcat/km

MX is a (what kind of inhibitor, the enzyme that is inhibitred)

reversible inhibitor of DHFR (dihydro-folate reductase. This enzyme is also important for purine and thymidine synthesis in cancer cells (enzyme function wasn't discussed in class)

Kcat can be duduced by looking at the "cat" word and realizing it is

showing how fast the enzyme is at converting substrate to product. This will happen when the enzyme complexes with the substrate.

Km: is aka

the Michael's Constant.

Vmax can also be thought of as

the maximal rate of the reaction that will be carried by the enzyme (wasn't discussed in class though)

Kcat is and what does it measure? (2 ways of stating)

the turnover number: 1) It measures how fast the E.S. complex breaks down to Enzyme and Product OR 2) When the enzyme is fully saturated, the number of moles of the substrate converted to product per mole of enzyme when it is fully saturated (in other words all the active sites are complexed with the substrate) per unit time

Low Km value means that: Explain

very good affinity between the substrate and enzyme - they bind tighly. For example, the substrate has high affinity to the enzyme that we don't need much of it to reach half of Vmax (and hence Vmax) faster.

Kcat / Km is, what do we use it for? Compare with Km.

(specificity constant); If you compare 2 substrates of the same enzyme and you want to say which substrate is a better substrate. It is more accurate than Km because it takes into account Km and Kcat.

How is the effect of a CI depicted on a LWB Plot (How is the effect of CI shown on LWP? What happens to Vmax (or 1/Vmax)?

1) CI = Competitive Inhibitor. 2) An increase in the Km (slope of CI will move to the right (on the x axis relative to the slope without the CI) However 3) The y intercept = 1/Vmax won't change because Vmax won't change and hence all the slopes (the slope without the CI + the slopes showing the CI at different concentrations) will have the same y intercept.

Malonate vs Mevalonate

1) In biosynthesis of cholesterol: 3 molecules of acetyl Coa will be converted to HMG-COA which will be converted by HMC-CoA reducatease to MEVALONATE 2) Malonate: Compeitive Inhibitor of SDH

Discuss the categorization/types of inhibitors we discussed so far (noncompetitive, irreversible, reversible and competitive inhibitors)

1) Two types of inhibitors: Reversible and Irreversible 2) Two types of Reversible inhibitors: Competetive and noncompetitive inhibitors.

On the LWB plot why does Km increase and Vmax stays the same (important)

1) When you measure Km you're at a low substrate concentration. Hence with CI, you'll need more substrate concentration to get to ½ Vmax because the inhibitor is interfering with the enzyme because we have low concentration of enzyme ( inhibitor is not diluted) = Lower Km. 2) When you measure Vmax, it is measured under inifinite concentrations of the Substrate. In other words all the enzymes are bonded to substrate (are in E.S. complex and none are available to bind to the inhibitor). In other words, we can perfectly achieve the same vmax by diluting the inhibitor with higher concentration of the substrate.

Describe what's on the LWB plot (what's on the x and y axes coordinates, what are the intercepts and what is the slope?)

1) on the x axis we have 1/[S] 2) on the y axis we have 1/V0 3) X intercept (when y = 0) = -1/Km (can be derived) 4) Y intercept (when x = 0) = 1/ Vmax 5) Slope is = Km/Vmax and its base will be at the x intercept.

i. Low Ki is (explain the reasoning why) ii. High Ki:

1) potent CI (competitive inhibitor); less potent CI. 2) Ki is a Keq and hence it is = Products over reactants. Therefore Keq or Ki = E + I / E.I. Higer E.I means lower Ki which explains why a low Ki indicates a potent inhibitor that has high affinity for the enzyme.

Km can reflect the different properties of isozymes. Explain with an example.

1. Hexokinase and glucokianse: both put phosphate group on glucose - that's why they're isozymes. 2. Hexokinase works in all the cells except the liver. 3. Glucokinase: present only in the liver. 3. Hexokinase's Km is about .155 mM (high affinity) and glucokinase is about 20 mM (lower affinity because higher Km) 4. In other words, Hexokinase will be quickly (readily) saturated because it has low Km and hence high affinity to sugar. 5. Glucokinase (in the liver) has high Km, you can NEVER saturate glucokinase (because it never gets saturated). This is a good thing because otherwise sugar will hang out in the blood and it It can do a lot of harm if it stays there. 6. The fact that hexokinases will get saturated faster will make all the sugar goes to the other enzyme which has low affinity to Km and will never get saturated. This is a great thing because you can always convert any excess sugar in the blood to glycogen and prevent it from doing harm if stays in the blood.

Properties of Competitive Inhibitors (very important)

1. Look like the substrate and hence competes with it for a spot at the active site. 2. Binds at Active Site. 3. However will not be processed by the enzyme to product 4. It just sits at the active site and blocks it.

Km can be used to measure: (2 things)

1. The affinities of different substrates to the SAME ENZYME to compare their binding strength 2. How the affinity of the solute (e.g. drug) to the enzyme changes after enzyme mutation.

The drugs (brand and generic) that inhibit HMG-CoA Reductase (competitive or non-competitve inhibitors, reversible or irreversible?

1. Zocor (Simvastatin) 2. Lipitor (Atorvastatin) (both are competitive reversible inhibitors) (see p.12 and then p.11 to see how they work)

If you want to measure Vmax, it will be

2 times V0 at Km.

In biosyntehsisi of cholesterol, 2. If you shut it down then you shut down the synthesis of cholesterol. b. Look at page 12: i. Now look at page 11.

3 molecules of acetyl Coa will be converted to HMG-COA which will be converted by HMC-CoA reducatease to Mevalonate

The 2 agents used in Cancer Chemotherapy.

5-Fluorouracil and Methotrexate.

5-FU

5-Fluorouracil. An enzyme inhibitor used in Cancer Chemotherapy to get rid of cancer cells.

Glucose metabolism in the cells.

All cells (except liver cells) will take the amount of sugar they need from bloodstream for fuel through hexokinase then the excess glucose will go to the liver and you can make glycogen.