The Queens College Guide to Life
M-M equation Vo = Vmax So L-B eqn: 1/Vo = Km/Vmax(1/So) + 1/Vmax The chemical basis of enzyme specificity is the complimentary relationship between. From these data, determine Vmax, KM, kcat, and the turnover number for the enzyme. From the intercepts in the plot: Vmax = 10 µM/min;. Km = 5 M = 5 µM;. But if a substrate has low Km, then it will also likely have low specific activity. There's no general relationship between the Km of an enzyme and the kcat.
Binding Energy contributes to reaction specificity and catalysis Specificity arises from the substrate needing to have its functional groups in certain areas to form hydrogen bonds and other weak interactions in the right places with the binding site. Some things that contribute to deltaGtrans, and form a barrier to reaction: The entropy of molecules in solution, which reduces the possibility that they will react together.
The solvation shell of hydrogen bonded water that surrounds and stabilizes solutes. The distortion of substrates that must occur in many reactions The need for proper alignment of catalytic functional groups on the enzyme Binding to an enzyme reduces entropy and thus increases the probability of the substrates reacting with each other. Reactants with restricted freedom of movement are limited to only a few conformations, some of which allow them to react.
Formation of weak bonds also results in desolvation of substrate, but the hydrogen bonds and weak interactions in the binding site replace the bonds with water that stabilized the substrate, so binding energy gets us out of this problem. Binding energy of reactions formed just in the transition state compensates for distortions the molecules have to go through in the reaction, like redistribution of electrons.
Induced fit postulated by Koshland allows the protein to take on a conformation that arranges its binding site functional groups into the right places to bind to the right spots of the substrates. Specific catalytic groups contribute to catalysis The binding energy from weak interactions is just one part of the catalytic mechanism.
Other forms of catalysis, general acid-base catalysis, covalent catalysis, and metal ion catalysis involve covalent bond formation, so are distinct from binding energy. General Acid-Base Catalysis charged intermediates in a reaction can be unstable and break down into reactants again.
To prevent this break down protons can be transferred to or from the reactant. This is done with substrates in enzymes. Sometimes the intermediate will stabilize itself by taking protons from water or donating to it via specific acid-base catalysis.
Most of the time you need general acid-base catalysis though, which is proton transfers involving non-water molecules. In our case, charged groups on amino acids in the binding site. Covalent Catalysis involves a transient covalent bond formed between enzyme and substrate.
Biochemistry quiz 5
This covalent bond can also activate a substrate for further reaction with the enzyme. Metal Ion Catalysis involves several possible ways a metal ion can interact with the substrate.
Enzyme bound metal groups can orient the substrate or stabilize charged transition states. Many enzymes use all of these. Chymotrypsin is an example. Enzyme Kinetics as an Approach to Understanding Mechanism Substrate concentration affects the rate of enzyme-catalyzed reactions Biologists look at the change in the rate of a reaction in response to various parameters.
This is enzyme kinetics. But it changes as the reaction proceeds. To simplify and account for this we use the initial rate V0. Since enzyme is present in nanomolar quantities, and [S] is much higher, changes in [S] at the beginning of a reaction are very small, and so [S] can be considered constant. Varying starting [S] concentration with [S] held constant affects V0: At low concentrations of [S], Vo increases almost linearly with increasing [S].
At higher concentrations, Vo increases by smaller amounts. Finally increases in Vo are vanishingly small with increasing [S].
Flashcards - Biochemistry quiz 5
This plateau region is close to the max velocity Vmax. Catalysis proceeds in two steps. The following is the fast step: The slower reaction is: This is the rate-limiting step. Thus the overall rate is proportional to the concentration of ES. At low concentrations of [S], most enzyme is uncombined E. The max initial rate of the catalyzed reaction Vmax is observed when almost all the enzyme is in ES form and [E] is tiny. Increases in [S] have no effect on rate. The concentrations close to Vmax are zero order kinetics.
In the beginning you have first order kinetics where rate is proportional to [S]. This change to zero order kinetics as S concentration increases is the saturation effect. The [S] at which the initial velocity v is at half Vmax is the michaelis constant Km. When the enzyme is first mixed into vastly more substrate, there is an initial period where ES slowly builds up pre-steady state.
This is too short to be observed. The reaction quickly goes into steady state where [ES] and the concentrations of all other intermediates remains almost constant over time. Measured Vo is usually the steady state, and analysis of these rates is steady state kinetics. The relationship between substrate concentration and reaction rate can be expressed quantitatively This graph looks the same for most enzymes.
It is the plot of the michaelis menten equation: So we note that we can represent free enzyme by subtracting the bound enzyme by the total: We also note that the amount of substrate bound is negligible compared to [S].
These two conditions let us derive Vo with more useful variables: Setting the two equations equal gives us: Distributing [S] and k1 on the left and taking out [ES] on the right: It is analogous to Kd.
Km has concentration units M. Note that when Vo is half Vmax: The shape of the curve is governed by these two simplified equations: Yo can use the Michaelis-Menten equation to calculate Km and Vmax via useful algebraic swapping. IT also describes inhibitor action as we will see later. Vmax is the theoretical maximum rate and is never obtained in reality. Reaching it requires all enzyme molecules bound. Kinetic parameters are used to compare enzyme activities All enzymes with a hyperbolic curve comparing Vo to [S] follow Michaelis-Menton kinetics.
But many enzymes that follow Michaelis-Menten kinetics do not have two step reactions like in steady-state kinetics, and have many different rates. So Vmax and Km do not provide much information about the number, rates, or chemical nature of steps in the reaction. But we use steady-state kinetics anyway. Interpreting Vmax and Km the curve in point 2.
This is obtained from the lineweaver-burk equation which results from taking the reciprocal of Vo in the Michaelis-Menten equation: This accurately determines Km, as opposed to approximates it like Vo versus [S] plots. Km can mean different things. Km measures the binding affinity here small Km means binding is tighter, large Km means binding is weaker. Km is thus not a simple measure of enzyme affinity. Even more complicated when reaction has several steps after ES formation.
Then Km is function of many different rate constants. Vmax also varies greatly between enzymes. If there are multiple partially rate limiting step kcat is a complex function of those rate constants. Comparing catalytic mechanisms and efficiencies Km is typically comparable to the cellular concentration of substrate.
An enzyme that acts on a substrate present in low concentrations has a lower Km tighter binding than an enzyme that acts only when substrate is more abundant.
This is the specificity constant or the catalytic efficiency. These enzymes are maximally efficient. Note that this limit can be reached with different values of kcat and Km. Many enzymes catalyze reactions with two or more substrates. Each substrate has a Km with the enzyme. There are two ways this reaction could proceed: Both substrates bind to the enzyme to form product this reaction can proceed in two ways, the ES1S2 complex formed is the ternary complex: One substrate can come in to form product 1, and the second can come in to form product 2 in this reaction S1 binding to the enzyme produces a change in the enzyme by transfer of a functional group to it, which later binds to S2 to form product.
This is a ping-pong or double-displacement mechanism. You can use a double-reciprocal plot to look at two-substrate reactions.
If the lines are parallel, the reaction uses a ping-pong pathway. If the lines intersect, the reaction forms a ternary complex. The pre-steady state allows us to measure these rates independently. This requires specialized rapid mixing and sampling techniques. Enzymes Are Subject to Reversible or Irreversible Inhibition Inhibitors are molecules that slow or halt enzymatic reactions.
There are two classes of inhibitors. Reversible inhibition includes, competitive, uncompetitive, and mixed inhibition.
Structural Biochemistry/Enzyme/Michaelis and Menten Equation
When I the inhibitor is bound, substrate cannot get in. Many competitive inhibitors are structurally similar to the substrate. We can analyze this with steady state kinetics. During inhibition the Michaelis-Menten equation becomes: Competition can be biased to favor substrate by adding more substrate. When [S] far exceeds [I] the reaction has a normal Vmax effect of I is negligible. Thus an increase in Km while Vmax does not change upon adding a compound shows it is a competitive inhibitor.
We will talk about these plots later. An example of competitive inhibition is ethanol used to treat patients that have ingested methanol. Alcohol dehydrogenase converts methanol to toxic formaldehyde.
This conversion is inhibited by ethanol. Technically ethanol is also a substrate for alcohol dehydrogenase and so the concentration will decrease over time, but it acts like a CI. Uncompetitive inhibitors bind at different sites from the active site, and bind only to the ES complex.
In uncompetitive inhibition the michaelis-menten equation is: Uncompetitive inhibitors thus lower measured Vmax. But it can bind to either E or ES. The michaelis-menten equation is: Mixed inhibitors affect Km and Vmax. We can use double-reciprocal plots to detect what kind of inhibition is going on. Uncompetitive and mixed inhibition are observed only for enzymes with two or more substrates. If an inhibitor binds to the site normally occupied by S1, it is a competitive inhibitor in experiments in which [S1] is varied.
If it binds to the site normally occupied by S2, it may act as a mixed or uncompetitive inhibitor of S1 because it binds to an alternative site from the binding site. The patterns observed depend on whether binding is ordered or random, so you can determine the order from observing them. Irreversible inhibition involves covalent binding or strong covalent association with the enzyme or destruction of a functional group on it.
Suicide inactivators are irreversible inhibitors that act like normal substrates but instead of being turned into product they turn into very reactive compounds that bind with the enzyme irreversibly.
They are also called mechanism-based inactivators for this reason. Enzyme Activity Depends on pH The pH range over which an enzyme is active can give a clue to the type of residue in the active site. A change in activity near pH 7. However, we must use caution. Within an enzyme the pKa of amino acid side chains can be very different from normal.
A positive charge near a Lys can lower the pKa of it significantly for example. The temporal sequence in which enzyme-bound reaction intermediates form The structure of each intermediate and each transition state The rates of interconversion between intermediates The structural relationship of the enzyme to each intermediate The energy contributed by all reacting and interacting groups to intermediate complexes and transition states.
We look in this section at chymotrypsin, hexokinase, enolase and lysozyme The Chymotrypsin Mechanism Involves Acylation and Deacylation of a Ser Residue Chymotrypsin cleaves peptide bonds by hydrolysis. It cleaves at Trp, Phe, and Tyr aromatics. Water does not directly attack the peptide bond, instead a transient covalent acyl-enzyme intermediate is formed.
Thus there are two phases: In the acylation phase the peptide bond is cleaved and an ester linkage is formed between the carbonyl of the peptide and the enzyme. In the deacylation phase, the ester linkage is hydrolyzed and the nonacylated enzyme is regenerated. This mechanism was proved by experimenting with the hydrolysis of p-nitrophenylacetate by chymotrypsin. It is measured by release of p-nitrophenol which is colored.Enzyme Kinetics (Km and Vmax) - Part 1
Initially the reaction releases a rapid burst of p-nitrophenol, nearly 1 molecule for each molecule of enzyme present. This is the fast acylation phase. Afterward the rate is slowed by the deacylation phase. On the left is a representation of primary structure, showing disulfide bonds and the amino acid residues crucial to catalysis. The protein consists of three polypeptide chains linked by disulfide bonds.
The active-site amino acid residues are grouped together in the three-dimensional structure. On the right is a close-up of the active site with a substrate mostly green bound. Two of the active-site residues, Ser and His57 both redare partly visible. This small Km will approach Vmax more quickly than high Km value. The enzyme efficiency can be increased as Kcat has high turnover and a small number of Km. Taking the reciprocal of both side of the Michaelis-Menten equation gives: To determined the values of KM and Vmax.
The double-reciprocal of Michaels-Menten equation could be used. Lineweaver-Burk graphs are particularly useful for analyzing how enzyme kinematics change in the presence of inhibitors, competitive, non-competitive, or a mixture of the two.
There are three reversible inhibitors: They can be plotted on double reciprocal plot. Competitive inhibitors are molecules that look like substrates and they bind to active site and slow down the reactions. Therefore, competitive inhibitors increase Km value decrease affinity, less chance the substrates can go to active siteand Vmax stays the same. Uncompetitive inhibitors can bind close to the active site but don't occupy the active site.
As a result, uncompetitive inhibitors lower Km increase affinity and lower Vmax. Non-competitive inhibitors are not bind to the active site but somewhere on that enzyme which changes its activity. It has the same Km but lower Vmax to those with no inhibitors. Km value is numerically equal to the substrate concentration at which the half of the enzyme molecules are associated with substrate.