MCAT Enzymes

What are the 6 major enzyme classes to know for the MCAT?

• Oxidoreductases → redox

• Transferases → transfer groups

• Hydrolases → break bonds using H₂O

• Lyases → add/remove groups to form double bonds

• Isomerases → rearrange within a molecule

• Ligases → join molecules using ATP

What do oxidoreductases do?

Catalyze oxidation-reduction (redox) reactions involving electron transfer.

Think: Oxidoreductase = REDOX

Examples: dehydrogenases and reductases.

What do transferases do?

Transfer a functional group from one molecule to another.

General idea:
A–B + C → A + B–C

Think: Transferase = transfers something

What do hydrolases do?

Break bonds using water.

A–B + H₂O → A–H + B–OH

Think: Hydro = water → water breaks it

What do lyases do?

Add or remove groups without using water, often creating or destroying a double bond.

Think: Lyase = leaves a double bond

What do isomerases do?

Rearrange atoms within the SAME molecule.

The molecular formula stays the same, but the structure changes.

Think: Isomerase → makes an isomer

What do ligases do?

Join two molecules together, usually using ATP.

A + B + ATP → A–B + ADP + Pi

Think: Ligase = links

Which enzyme class typically uses ATP to join two molecules together?

Ligase

Ligases use ATP energy to create a new bond between molecules.

What does a kinase do?

A kinase adds a phosphate group to a molecule, usually by transferring the phosphate from ATP.

ATP → ADP

Think: Kinase = puts phosphate ON

Are kinases transferases?

YES.

Kinases are transferases because they transfer a phosphate group from ATP to another molecule.

What is the general kinase reaction?

ATP + substrate → ADP + phosphorylated substrate

The substrate gains a phosphate.

What does a phosphatase do?

A phosphatase removes a phosphate group using water.

Think: Phosphatase = phosphate OFF

What enzyme class are phosphatases?

Hydrolases

They use H₂O to hydrolyze/remove the phosphate group.

Kinase vs phosphatase?

Kinase → phosphate ON
Phosphatase → phosphate OFF

Kinase usually uses ATP.
Phosphatase uses H₂O.

Does phosphorylation always activate an enzyme?

NO.

Phosphorylation can activate OR inhibit a protein depending on the protein.

Do not automatically assume phosphate = activation.

What does Km tell you?

Km reflects substrate concentration needed to reach ½ Vmax.

For typical Michaelis-Menten enzymes:

↓ Km = ↑ apparent substrate affinity
↑ Km = ↓ apparent substrate affinity

Think: Low Km = enzyme grabs substrate easily.

What does Vmax represent?

The maximum reaction rate when the enzyme is saturated with substrate.

All available active sites are essentially occupied.

What does a LOW Km generally mean?

Higher apparent affinity for substrate.

The enzyme reaches ½ Vmax with less substrate.

Low Km = loves substrate

What does a HIGH Km generally mean?

Lower apparent affinity for substrate.

More substrate is needed to reach ½ Vmax.

Where does a competitive inhibitor bind?

Free enzyme (E), at the active site.

It competes with substrate for the active site.

What happens to Km and Vmax with competitive inhibition?

Km ↑
Vmax SAME

Why does Km increase in competitive inhibition?

The inhibitor competes with substrate, so more substrate is required to reach ½ Vmax.

Therefore apparent affinity decreases → Km ↑

Why does Vmax stay the same with competitive inhibition?

Enough substrate can outcompete the inhibitor.

At very high substrate concentration, the enzyme can still reach the original Vmax.

Can competitive inhibition be overcome by increasing substrate concentration?

YES.

This is why Vmax remains unchanged.

Where does a pure noncompetitive inhibitor bind?

It binds E and ES equally at an allosteric site.

What happens to Km and Vmax with pure noncompetitive inhibition?

Km SAME
Vmax ↓

Why does Vmax decrease with noncompetitive inhibition?

The inhibitor effectively reduces the amount/activity of functional enzyme.

Adding more substrate cannot overcome it.

Why does Km stay the same in pure noncompetitive inhibition?

The inhibitor binds E and ES equally, so the enzyme's apparent affinity for substrate does not change.

Where does an uncompetitive inhibitor bind?

ES complex ONLY.

The substrate must bind first.

Think: UNcompetitive = UNder the substrate

What happens to Km and Vmax with uncompetitive inhibition?

Km ↓
Vmax ↓

Both go DOWN.

Why does Km decrease in uncompetitive inhibition?

The inhibitor binds and stabilizes the ES complex, making the enzyme appear to have greater substrate affinity.

Where does a mixed inhibitor bind?

Both:

E + ES

but UNEQUALLY.

What always happens to Vmax with mixed inhibition?

Vmax ↓ ALWAYS.

What happens to Km with mixed inhibition?

Km can ↑ OR ↓, depending on whether the inhibitor prefers E or ES.

Mixed inhibitor prefers FREE ENZYME (E). What happens?

Km ↑
Vmax ↓

Preference for E makes substrate binding harder → apparent affinity ↓ → Km ↑.

Mixed inhibitor prefers ES complex. What happens?

Km ↓
Vmax ↓

Preference for ES stabilizes ES → apparent affinity ↑ → Km ↓.

What is the relationship between mixed and noncompetitive inhibition?

Pure noncompetitive inhibition is a special case of mixed inhibition.

Mixed → binds E and ES unequally
Noncompetitive → binds E and ES equally

Memorize the Km/Vmax effects of the four major inhibitors.

Inhibitor	Km	Vmax	Binds
Competitive	↑	SAME	E only
Noncompetitive	SAME	↓	E + ES equally
Uncompetitive	↓	↓	ES only
Mixed	↑ or ↓	↓	E + ES unequally

Fastest memory:
Competitive = Km up
Noncompetitive = Vmax down
Uncompetitive = both down
Mixed = Vmax down, Km moves

What are the axes of a Lineweaver-Burk plot?

Y-axis = 1/V

X-axis = 1/[S]

What is the y-intercept of a Lineweaver-Burk plot?

1/Vmax

Therefore:

Y-intercept ↑ → Vmax ↓

What is the x-intercept of a Lineweaver-Burk plot?

−1/Km

Important MCAT shortcut:

Left = Km ↓
Right = Km ↑

What is the slope of a Lineweaver-Burk plot?

Km/Vmax

What is cooperativity?

When substrate binding at one site affects substrate affinity at other binding sites on the same protein/enzyme.

Usually occurs in proteins with multiple subunits/binding sites.

What is positive cooperativity?

Binding of one substrate INCREASES affinity for subsequent substrate molecules.

First binds → next binds easier

What is negative cooperativity?

Binding of one substrate DECREASES affinity for subsequent substrate molecules.

First binds → next binds harder

What is noncooperative binding?

Binding of one substrate does NOT affect the affinity for subsequent substrates.

What type of curve is associated with positive cooperativity?

Sigmoidal (S-shaped) curve

Classic example: hemoglobin binding O₂.

Michaelis-Menten enzymes vs cooperative enzymes: curve shape?

Michaelis-Menten → hyperbolic

Positive cooperativity → sigmoidal/S-shaped

Where does glycolysis occur?

Cytosol

What is the purpose of glycolysis?

Break down glucose → pyruvate while producing ATP and NADH.

What are the two major phases of glycolysis?

1. Investment phase → spend ATP

2. Payoff phase → produce ATP + NADH

Think: Spend money first → make money later.

How many ATP are invested in glycolysis?

2 ATP

How many ATP are produced in glycolysis?

4 ATP gross

Because 2 ATP were spent:

Net = 2 ATP

What is the NET yield of glycolysis per glucose?

2 pyruvate
2 ATP net
2 NADH

Which glycolysis enzymes consume ATP?

Hexokinase
Phosphofructokinase-1 (PFK-1)

Both are kinases.

What does hexokinase do in glycolysis?

Glucose → glucose-6-phosphate

Uses:

ATP → ADP

Hexokinase adds a phosphate to glucose.

What does phosphofructokinase-1 (PFK-1) do?

Fructose-6-phosphate → fructose-1,6-bisphosphate

Uses:

ATP → ADP

PFK-1 is the major rate-limiting/regulatory enzyme of glycolysis.

What is the rate-limiting enzyme of glycolysis?

Phosphofructokinase-1 (PFK-1)

⭐ Very high-yield MCAT fact.

Which glycolysis step produces NADH?

Glyceraldehyde-3-phosphate dehydrogenase

NAD⁺ → NADH

Because it's a dehydrogenase, think redox.

What type of enzyme is a dehydrogenase?

Oxidoreductase

Dehydrogenases participate in redox reactions involving electron/hydrogen transfer.

Which glycolysis enzymes directly produce ATP?

Phosphoglycerate kinase
Pyruvate kinase

They perform substrate-level phosphorylation.

What is substrate-level phosphorylation?

Making ATP by directly transferring a phosphate from a metabolic intermediate to ADP.

ADP → ATP

Occurs in glycolysis.

Why can a kinase sometimes PRODUCE ATP instead of consuming it?

Kinases transfer phosphate groups.

The phosphate does NOT always have to come from ATP.

For example, pyruvate kinase transfers phosphate to ADP, producing ATP.

⭐ So remember:

Kinase = phosphate TRANSFER, not simply "uses ATP."

What does pyruvate kinase do?

Phosphoenolpyruvate (PEP) + ADP → pyruvate + ATP

It produces ATP through substrate-level phosphorylation.

What are the enzyme facts I absolutely need memorized for the MCAT?

Oxidoreductase = redox
Transferase = transfers groups
Hydrolase = breaks using H₂O
Lyase = add/remove → double bonds
Isomerase = rearranges
Ligase = links molecules + ATP

Kinase = transfers phosphate
Phosphatase = removes phosphate

Competitive: Km ↑, Vmax same
Noncompetitive: Km same, Vmax ↓
Uncompetitive: Km ↓, Vmax ↓
Mixed: Vmax ↓, Km either way

Positive cooperativity: binding → next binding easier
Negative cooperativity: binding → next binding harder

Glycolysis: glucose → 2 pyruvate + 2 ATP net + 2 NADH

PFK-1 = rate-limiting enzyme of glycolysis.