Enzymes Flashcards
Oxioreductases
catalyze oxidation-reduction reactions; transfer e-
- need NAD+ cofactor
Transferases
catalyze transfer of C, N, or P- containing groups
- need THF cofactor
Hydrolases
catalyze cleavage of bonds by addition of water
Lyases
catalyze cleavage of CDC, CDS and certain CDN bonds
- no H2O
Isomerases
catalyze racemization of optical or geometric isomers
- transfer within the same molecule
Ligases
catalyze formation of bonds between C and O, S, N coupled to hydrolysis of high energy phosphates
- makes new bonds
- ONLY makes something bigger
Cofactor
metal ions
Coenzymes
small organic molecules, mostly derived from vitamins
holoenzyme
apoenzyme + cofactor/coenzyme = holoenzyme
- apoenzymes without cofactors are inactive
- most cofactors are regenerated at the end of the reaction
Enzymes
- biological catalysts
- highly specific
- extremely fast
- activity can be regulated
Enzymatic Reactions Multistep
1) enzyme binds to substrate
2) ES->EP
3) dissociation of EP to P and regeneration of E
E + S ES E + P
Catalyst
- REGENERATED at the end of reaction
- accelerates reaction
- does not change spontaneity
Catalytic Amino Acids/Active Site
scaffold creating active site; amino acids close together in tertiary structure but not in primary structure
- NEED to maintain structure to maintain active site
Enzyme Active Site
- substrates and products bind reversibly through weak noncovalent interactions; numerous weak interactions lead to tight enzyme substrate bonding
- small volume compared to all of enzyme
- generally nonpolar (helps increase interactions)
Non covalent Interactions:
- electrostatic: ionic, dipole-dipole
- Hbonds
- Hydrophobic
Denature Proteins
via high temperature and acid; unfolds proteins and effects active sites
- optimal temperature; change in rate is a bell curve
- optimal pH functionally specific
pH’s effect on enzymes
- at extremes of pH: irreversible denaturation
- at moderate pH: change of charge of enzyme functional groups can affect activity; this is reversible
Glucokinase/Hexokinase
precise active site conformation explains specific binding and reaction of ATP with GLUCOSE but NOT galactose
Lock and Key Inadequacies
- according to lock and key the active site should be able to accommodate smaller substrates, this is not the case
Induced Fit Model
FLEXIBLE active site; conformational change stabilizes active conformation to substrate after binding
- explains REGULATION and COOPERATIVE effects
Enzyme rate increases
10^6-10^17 fold
Relationship of reaction rate and activation energy
reaction rate is inversely proportional to activation energy
How do enzymes increase reaction rate
decrease the activation barrier by stabilizing the transition state
How do enzymes lower the transition state energy?
tighter binding of the active site amino acid residues to the transition state
Exergonic Reaction
spontaneous reaction
G<0 (negative)
Endergonic reaction
non spontaneous reaction
G>0 (positive)
Catalytic Strategies
- Proximity (ALL)
- Transition state stabilization (ALL)
- Covalent catalysis or nucleophilic catalysis (some)
- general acid-base catalyst (most)
- metal ion catalyst (many)
Proximity Effect
PROXIMITY AND ORIENTATION
increases the effective concentration of substrates; corrects orientation of substrates for efficient reaction
Protease enzyme
1) Active site- general acid-base catalysis
2) transition state stabilization (oxyanion hole)
3) covalent catalysis
Chymotrypsin
Active site - catalytic triad - serine, histidine, and aspartate (all serine proteases have catalytic triad)
- His accepts proton from serine to become HisH+
- serine becomes negatively charged - a potent nucleophile
- enzyme stabilizes negative charges in transition state b/c serine becomes oxyanion hole; activation energy decreased
Enzyme Active Site is Complementary to
the transition state structure rather than the substrate
Oxyanion hole
space in the enzyme active site ready to bind a negatively charged group
- serine stabilizes transition state
Enzyme Regulation with Extracellular Signaling
V Slow
Enzyme regulation
- feedback inhibitors, enzyme inhibitors, product inhibition, feedback activation
- enzyme CONCENTRATION changes– synthesis (trascrip, translat) and degradation
- compartmentation
- post-translational modifications
- regulatory proteins to activate or inhibit
Most common post-translational modification
phosphorylation
Phosphorylation of these amino acids can activate or inhibit an enzyme
ser, thr, tyr
Zymogens
inactive form of enzyme; regulated by specific protein clevage; irreversibly turn on, degraded when finished
Enzymes 3 main affects
- how tightly substrates bind
- how fast/what rate
- how regulated
kcat
rate product is made
- turnover number
- effected by pH and temp
rate S -> P
Km
equilibrium constant
- how tightly substrate is bound to enzyme
- [S] at 1/2 Vmax
- ** Km big = weaker affinity, bound and fell off; Km small = stronger affinity/tighter binding
Vmax=
total output; ~ complete saturation
Vmax=kcat x [E]
How do we measure initial velocity?
SLOPE!
Michaelis-Menten Equation
v= Vmax [S] / Km + [S]
Lineweaver-Burk Plot intercepts
x intercept = -1/Km
y intercept = 1/Vmax
Slope = Km/Vmax
What changes Km values
change with reaction conditions; pH or temp
Vmax is linearly dependent on
enzyme concentration
- more enzyme, higher rates
- is affected by pH and temp
kcat equation
kcat = Vmax/[E]
- kcat is normalized to [E]
- how fast EACH enzyme is producing output
- PER ENZYME
kcat Vmax equation
kcat x [E] = Vmax
Catalytic Efficiency
kcat/Km
- ratio of enzyme’s kcat and Km calues
- shows
1) enzyme’s substrate preference
2) enzyme’s catalytic efficiency
3) high the kcat/Km, the better the substrate and better the catalytic efficiency of the enzyme
Enzyme Inhibition
depends on active site
** molecules that resemble substrate or transition state structures without reacting are potential drugs **
2 types of inhibitors
Irreversible inhibitors (covalent drugs): molecules bind covalently to enzyme to inhibit activity Reversible inhibitors: molecules that bind reversibly to inhibit enzyme activity
Competitive Inhibitors
bind to same active site; competes with substrate binding; generally look alike
Km increased, Vmax unchanged
Noncompetitive Inhibitors
bind to a separate site from the active site; does not compete with substrate binding
Vmax decreased, Km unchanged
As lines move towards zero on a LWB plot
denominator gets bigger
Transition state analogs
competitive inhibitors
- stable molecules with geometric and/or electronic features of the highly unstable transition state
Feedback regulation
non-competitive
Allosteric Enzymes
- have an active site and an allosteric site
- oligomeric
- active site binds S and allosteric binds E
- ALLOSTERIC MOLECULES DO NOT RESEMBLE THE SUBSTRATE
Allosteric Enzyme kinetics
cooperative binding
- S shaped kinetics curve
- hard for first to bind, easy for following to bind
R-state vs T-state
- cooperative binding equilibrium conformations
- R: active; binds S better and has high catalytic activity
- T: inactive
Allosteric Activator
- stabilizes the R-state; increases S binding/activity
- shifts kinetics chart left
- both Km and Vmax are affected
Allosteric Inhibitor
- stabilizes the inactive T-state; decreased S binding/activity
- shifts kinetics curve right
- both Km and Vmax are affected
Allosteric vs Noncompetative
Noncompetitive: synthetic drug
Allosteric: actual molecule in cell
Aspartate Transcarbamylase
conformationally changes and cooperative binding
Isozymes
- have different primary structures of AA sequence, but catalyze the same chemical reaction and act upon the same substrates
- distinct expression in different tissues of the body
- allow find tuning of metabolism to meet the needs of a given tissue or developing stage
- have different kcat and Km values and different temperature and pH dependencies
- thought to have evolved from gene duplication and divergence
Biomarkers
measurement of isozyme levels helps in diagnosis
