enzyme kinetics Flashcards
(34 cards)
Catalyst
a compound that increases the rate of a chemical reaction (e.g. making and breaking covalent bonds) without being changed in the process. Thus, enzymes accelerate specific reactions needed in a biological system. They do so by forming a specific three-dimensional structure with an active site.
Enzyme characteristics
They are (primarily!) proteins with specific structures and active sites formed when the protein folds into its three dimensional shape. The formation of a highly ordered active site makes the enzyme very specific for its substrates. Some use specific cofactors or coenzymes. Coenzymes provide chemical groups for the reaction and are “used up.” Cofactors are not. Enzymes are classified and named by what they do.
cofactor
a non-protein chemical compound that is required for the protein’s biological activity. These proteins are commonly enzymes, and cofactors can be considered “helper molecules” that assist in biochemical transformations.
coenzyme
a complex organic or metalloorganic cofactor, most of which are derived from vitamins and from required organic nutrients in small amounts. Metabolism involves a vast array of chemical reactions, but most fall under a few basic types of reactions that involve the transfer of functional groups. These group-transfer intermediates are the loosely bound organic coenzymes. Each class of group-transfer reaction is carried out by a particular cofactor, which is the substrate for a set of enzymes that produce it, and a set of enzymes that consume it. An example of this are the dehydrogenases that use nicotinamide adenine dinucleotide (NAD+) as a cofactor.
prosthetic group
A coenzyme or cofactor that is tightly bound to the enzyme
holoenzyme
An inactive enzyme without the cofactor is called an apoenzyme, while the complete enzyme with cofactor is called a holoenzyme.
reaction equilibria
DG’o =-RTlnKeq When the energy of product is lower than substrate,
S will spontaneously convert into P. However, deltaG’° says nothing about the
rate at which this occurs.The rate of the reaction is dependent upon the activation energy, deltaG‡
Transition State (TS) Theory
there is a single path (sequence of chemical structures) for S to convert to P, and the TS is the highest energy structure along this path. The TS is equally likely to either proceed to form P or decay back to form S. The TS’s existence is very short lived – on the order of a molecular bond vibration. Thus, the rate of a reaction depends on the rate at which the ground state structures can form the TS. It has nothing to do with deltaG°’, rather, it is related to deltaG‡.
How enzymes work
The enzyme has decreased DG‡ (stabilized the transition state). Thus, the energetic “barrier” between the substrate and products is lower. This accelerates the RATE of the reaction. But, the equilibrium of the reaction HAS NOT CHANGED.
rate constant (k)
k = (κT/h) e-DG‡/RT where κ= Boltzmann constant h = Planck’s constant T = temperature. Therefore, since Velocity (V) = k [S], as the rate constant increase, the velocity of the reaction increases
How do enzymes lower DG‡?
- Binding energy between enzyme (E) and substrate (S) to form the ES complex. Multiple, weak noncovalent interactions that provide specificity as well as catalysis. Enzymes are optimized to bind the transition state. 2. Increase “local concentration” of substrates. 3. Rearrangement of covalent bounds in specific active site chemistry. (eg acid-base, covalent, and metal ion catalysis)
how enzymes stabilize the transition state structure
Enzyme active sites make specific non-covalent interactions with the substrate (hydrogen bonding, salt bridges, hydrophobic, etc.). These specifically interact with the transition state.
How enzymes increase “effective concentration”
The ES complex is essentially a single unit rather than a sum of many interactions as might occur in bulk solution. Both are entropically unfavorable, but a pre-formed active site is less so. Further, the active site orders multiple substrates, thus increasing their “effective concentration.” The active site can bring together multiple substrates, therefore greatly increases reaction probability
acid-base catalysis
Proton donors and acceptors, i.e. acids and base may donate and accept protons in order to stabilize developing charges in the transition state.This typically has the effect of activating nucleophile and electrophile groups, or stabilizing leaving groups.
Covalent catalysis
Covalent catalysis involves the substrate forming a transient covalent bond with residues in the active site or with a cofactor. This adds an additional covalent intermediate to the reaction, and helps to reduce the energy of later transition states of the reaction. The covalent bond must, at a later stage in the reaction, be broken to regenerate the enzyme.
Metal ion catalysis
A metal such as Cu2+ or Zn2+ can also stabilize the TS. The metal must be able to be bound to the charged intermediate and hence the TS. The tetrahedral oxyanion intermediate of the reaction of an electrophilic carbonyl C can interact with a metal if there is an O on an adjacent atom which can help coordinate the metal ion. This charge stabilization of the developing negative in the TS and the full negative in the intermediate is often called electrostatic catalysis. This method is likely to be found in many enzymes since nearly 1/3 of all enzymes require metal ions.
Measuring enzymatic parameters
step 1: Measure initial velocity, Vo, at one [S] step 2: Repeat your measurement at many [S] step 3: Create Vo vs. [S] plot
Km
the substrate concentration at which the reaction rate is half of Vmax. The Km is not the same as a binding constant (Kd). The Km can sometimes be a reflection of the binding affinity of E towards S. Indeed, typically Km values are approximately equal to intracellular S concentrations. But this is not always the case
Vmax
the maximum rate achieved by the system, at maximum (saturating) substrate concentrations.
Michaelis–Menten kinetics
V=[Vmax [S]]/[[S] + Km ]
kcat
the rate limiting step of any enzyme catalyzed reaction. kcat is the “turnover number” for an enzyme. It measures the number of substrate molecules turned over (into product) per enzyme molecule, per second. If several rate constants are partially rate limiting (generally, within an order of magnitude of the slowest rate constant), then kcat does not correspond to any single rate constant, but is a composite rate constant. The inverse is the amount of time for each subsrate molecule to be processed
kcat/Km
is a useful way to compare enzymes. the larger this number, the more efficient this enzyme
Reversible inhibition
types include: competitive: Bind only to E (enzyme without substrate). Uncompetitive: Bind only to ES complex. Mixed: Bind to both the E and ES complex. noncompetitive inhibitors are a type of mixed.
competitive inhibition
the substrate and inhibitor cannot bind to the enzyme at the same time, as shown in the figure on the left. This usually results from the inhibitor having an affinity for the active site of an enzyme where the substrate also binds; the substrate and inhibitor compete for access to the enzyme’s active site. This type of inhibition can be overcome by sufficiently high concentrations of substrate (Vmax remains constant), i.e., by out-competing the inhibitor. However, the apparent Km will increase as it takes a higher concentration of the substrate to reach the Km point, or half the Vmax. Competitive inhibitors are often similar in structure to the real substrate. kcat is unaffected while the apparent Km increases as [I] increases. Therefore Km changes but Vmax does not change
