Enzymes & Biological Catalysis
Enzymes Increase Reaction Rates
uncatalyzed
Moving Object ant crawling person walking race horse gallop Bugatti Veyron FA-22 Raptor Space shuttle Photon from sun Speed (mph) 0.06 3 40 253 1500 17,500 6.7 x 108
catalyzed
Rate Increase 1 101.6 102.9 103.7 104.4 105.5 108.8
Enzymes enhance reaction rates 5-17 orders of magnitude
Enzymes Select Specific Pathways
Enzymes catalyze one out of many possible reactions
O
acetaldehyde
B
O
CO2 ADP Pi HCO3ATP NADH pyruvate NAD+
OH
A E
-OOC
oxaloacetate
COO-
COO-
NAD+ NADH
O
CoASH CO2
lactate
COO-
acetyl-CoA
SCoA
4 possible reactions for pyruvate
Enzymes Allow Reaction Regulation
O
-OOC
ADP Pi
COO-
HCO3ATP
oxaloacetate
Enzyme-catalyzed reactions are subject to strict regulation
pyruvate pyruvate carboxylase CoASH NAD+ pyruvate dehydrogenase NADH CO2
O
COO-
SCoA
acetyl-CoA
OH
-OOC
COOCOO-
citrate
Enzyme Classes
Cofactors
Mg2+ Zn2+ Cu2+ Fe3+ Ca2+ bind enzyme transiently stably bound to enzyme
Vitamin-derived Coenzymes
Enzyme Active Sites
Active site: special environment that accelerates chemical transformations
Active Sites Bind Substrates
Reaction Equilibria and Free Energy
[B] K eq = [A]
A B
G = RT lnK eq
A quantitative measure of the tendency of a reaction
28
Mix A, B
Measure A, B
G > 0, reaction unfavored (endergonic) G < 0, reaction favored (exergonic) G = 0, reaction at equilibrium
G 0
-28 10-5 1 105
Keq
Effect of Enzymes
Enzymes Lower Energy of Transition State
Enzymes Lower Energy of Activation
Enzyme binding to substrate and product critical for rate enhancement
The ES Complex Weak bonds bind substrate to enzyme.
Provides much of energy needed to lower G. ES formation accompanied by lower G, termed binding energy (GB).
Major source of energy used to lower G.
GB contributes to specificity and catalysis. Weak interactions between E and S are optimized at the transition state.
Active sites shape is complementary to the geometry of the reaction transition state.
Formation of ES Complexes
Lock-and-key model
Induced-fit model
Summary of Key Points
Geometry and chemical make-up of active site responsible for rate enhancement and specificity
Enzymes greatly accelerate reactions in a highly specific manner
Acceleration due to reduction in energy needed to reach transition state NO effect on Keq
Favorable weak interactions between E and S promote ES, lower transition state energy
Maximal interactions between E and S occur when S assumes the shape of the transition state
Enzyme Kinetics
[Vmax ][S] v0 = K m + [S]
Michaelis-Menton equation
(when Km >> [S]) (when [S] >> Km)
(when Km = [S])
Double-reciprocal Plot Estimates Vmax, Km
Turnover Number: kcat
Estimates the rate an enzyme can operate on a per active site basis under conditions when the enzyme is saturated
kcat = maximum number of molecules of substrate that can be converted to product per second per active site
V max k cat = ; [ET]
kcat[ET][S] Vo = Km + [S]
kcat units: s-1
Specificity Constant: kcat/Km Under physiologic conditions, most enzymes operate at [S]:Km = 0.01-1.0.
If Km >> [S], then Specificity constant measures speed of enzyme when [S] low.
Index of efficiency of intermolecular collisions.
Diffusion controlled reactions have k~108 .
Vmax[S] kcat v = [E][S] Km Km
Units: M-1s-1
Allows v comparisons of different substrates for a given enzyme.
Enzyme Inhibition
Various agents block enzyme activity
Ibuprofen
O
OH
Types of Inhibition
Irreversible inhibitors Reversible inhibitors Competitive Uncompetitive Noncompetitive
Form irreversible complex with E, mechanism-based inactivators
(aka, noncompetitive)
Competitive Inhibition
v0 =
Vmax [S] K M + [S]
= 1+
[I] KI
is index of inhibitors affinity for E
Uncompetitive Inhibition
Vmax [S] v0 = K M + a [S] [I] = 1+ KI
is index of inhibitors afnity for ES complex
Noncompetitive (Mixed) Inhibition
Vmax [S] v0 = K M + [S]
Table 12-2
Effect of Ibuprofen on PGG2 [S] 0.5 1.0 1.5 2.5 3.5 Vo 23.5 32.2 36.9 41.8 44.0 +IB 16.67 25.25 30.49 37.04 38.91 1/[S] 2 1 0.67 0.40 0.286 1/Vo 0.043 0.031 0.027 0.024 0.023 +IB 0.06 0.04 0.033 0.027 0.026
Catalytic Strategies
Promote favorable geometric configuration and proximity of substrates Guiding principles Enhance partial charges on reactive species Stabilize transition states Stabilize charged reaction intermediates
Proximity and Orientation Effects
Enzymes bring reacting species close together. Energy of interaction helps overcome entropy decrease. Reactants bound with orientation that promotes transition state.
GAPDH in complex with NAD+ and GAP (PDB 1NQO)
O P -O OOH O H O
glyceraldehyde-3-phosphate NAD+, Pi
NADH
O P -O OOH O O OO O P O-
1,3-diphosphoglycerate
Intramolecular v. Intermolecular Reaction Rates
Intramolecular reactions faster
General Acid, General Base Catalysis
Bond rearrangement or breakage requires electron migration Reactive chemical groups: electrophiles, nucleophiles
O R' O
O R' O O C R HO R' H
O C R
C + R
H H
OH
General Acid Catalyzed Reactions
General Base Catalyzed Reactions
Electrostatic Effects
A xed charge or polar group can stabilize the transition state.
Positive charged moiety can stabilize transient negative charges.
O C R1 O R2 R1 O H O H R3 O C O R3
NH3 OC O R2
Active sites often hydrophobic.
Low dielectric constant enhances charge on ionic groups, increases electrostatic interactions.
tetrahedral transition state
Enzymatic Nucleophiles, Electrophiles
A group on an enzyme acts as a nucleophile or electrophile
Structural Flexibility
Substrate binding often induces conformational change in enzyme. Overall effects:
Examples
Hexokinase
Glc + ATPGlc-6P + ADP
Chymotrypsin
HK
HK + Glc
Serine Proteases
Large family of hydrolases with diverse functions
O O N+ H2O
acetylcholine
acetylcholinesterase
O OHO N+
Catalytic triad: Asp, His, Ser
His
acetate
choline
Asp O OH N N H O Ser
Active Site is 3-Dimensional Construct
Asp, His, Ser are not contiguous
Triad only comes together when protein folds into native structure
Trypsin
Specificity Pocket
Protease chymotrypsin trypsin Elastase
Specificity Phe/Trp/Tyr-X Lys/Arg-X small neutral (Ala)-X
General Serine Protease Mechanism
Figure 11-29 part 5
Zymogens
Proteolytic enzymes usually synthesized as inactive precursors Zymogenic form prevents active proteases from digesting tissues that synthesize them
pancreatic acinar cells
Trypsinogen: Zymogen of Trypsin
trypsinogen-specific peptide signal sequence active trypsin
MKTFIFLALL GANTVPYQVS AAHCYKSGIQ SKSIVHPSYN SRVASISLPT GTSYPDVLKC SNMFCAGYEG GIVSWGSGCA TIASN
GAAVAFPVDD LNSGYHFCGG VRLGEDNINV SNTLNNDIML SCASAGTQCL LKAPILSDSS GKDSCQGDSG QKNKPGVYTK
DDKIVGGYTC SLINSQWVVS VEGNEQFISA IKLKSAASLN ISGWGNTKSS CKSAYPGQIT GPVVCSGKLQ VCNYVSWIKQ
Enteropeptidase or trypsin clips off VDDDDK peptide, activating trypsin
Trypsin Activation by Ile 16
N-term Ile NH2 bonds to Asp 194
Frees up active site to bind substrate
