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Phosphoryl Transfer. In biological systems, the element phosphorous almost always exists as phosphate. Phosphorous is stable in several different oxidation states, but in phosphate, the oxidation state is +5. Therefore, the phosphorous atom in phosphate will always behave as an electrophile.
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Phosphoryl Transfer • In biological systems, the element phosphorous almost always exists as phosphate. Phosphorous is stable in several different oxidation states, but in phosphate, the oxidation state is +5. Therefore, the phosphorous atom in phosphate will always behave as an electrophile. • Phosphorous can form more than four covalent bonds. As a second-row element, it has low lygin d orbitals into which additional electron pairs can be put to form a fifth bond. In the phosphate group, the unshared electron pair on one of the oxygen atoms can be shared with a d orbital of the phosphorous to form a d-pp bond.
Classes of Phosphoryl Transfer In kinases, X is almost always ADP. However, GDP is known to substitute i some cases.
Kinases • Kinases are phosphotransferases that catalyze the transfer of a phosphoryl group to an acceptor molecule. Most often, the phosphoryl group comes from the terminal (gamma) position of adenosine triphosphate. • There is high negative charge associated with the triphosphate group of ATP, which shields each phosphorus against reaction with incoming nucleophiles. This property makes ATP kinetically stable int he cell, although thermodynamically, its hydrolysis is favorable. In enzyme catalysis, these charges are typically neutralized in order to facilitate nucleophilic attack. • Coordination with metal ions. Most often magnesium. In the cell, ATP is frequently found associated with magnesium, and the true substrate is MgATP. • Ion pairing with positively charged amino acids such as the guanidinium of arginine, or the lysine ammonium group. • From the structure of ATP, chemical precedent would indicate that the g-bond would be cleaved via a dissociative transition state, while the a and b-bonds would be cleaved via associative transition states.
Adenylate Kinase • The transfer of phosphoryl groups between different nucleotides, as well as other small molecules is important for utilizing and replenishing the cellular pool of energy-rich phosphate compounds. Adenylate kinase is a classic example of enzymes in this class. • Adenylate kinase was formerly known as myokinase because it is found in high concentrations in muscle tissue. • The adenylate kinase reaction is isoenergetic. A phosphoanhydride is cleaved and formed on both sides of the equation. • Adenylate kinase displays sequentialkinetics, in which both substrates must be bound before any product is released. • This is distinguished from what is termed ping-pong kinetics, in which one reactant modifies the enzyme, and then a second reactant interacts with the modification.
Bi-substrate Enzyme Kinetics Sequential 1.ordered 2.random Ping-pong
Equations for Bi-substrate Kinetics [B] 1/v Vmax[A][B] v = Ka[B] + Kb[A] + [A][B] 1/[A] [B] Vmax[A][B] 1/v v = [A][B] + Ka[B] + Kb[A] + KaKb 1/[A]
Sequential Kinetics • Sequential kinetics can be distinguished from ping-pong kinetics by initial rate studies. • In practice, measure initial rates as a function of the concentration of one substrate while holding the concentration of the second constant. Next, vary the concentration of the second substrate and repeat. • Lineweaver-Burk (double-reciprocal) analysis should yield a family of lines that intersect at the left of the y-axis of the graph. • Within the realm of sequential reactions lies ordered sequential and random sequential at the extreme ends. The equations for the two are identical; therefore, simple initial rate studies cannot differentiate between the two. • In ordered sequential reactions, one substrate is obligated to bind to the enzyme before a second substrate. In random sequential mechanisms there is no preference. In practice, there is usually some degree of order in binding.
Adenylate Kinase Kinetic Pathway Adenylate kinase displays a random ordered kinetic mechanism. In this case, the two substrates are bound randomly, and are in equilibrium with the “ternary complex” (E•MgATP•AMP). As in our derivation, this necessitates that the off rate for each of the substrates is less than the forward rate constant for the chemical step. This allows us to replace Km with Ks. However, it would not be incorrect to use Km values. Below is typical shorthand notation for kinetic schemes.
Nucleoside Diphosphate Kinase • Nucleoside diphosphate kinase (NDP Kinase) catalyzes the transfer of the terminal phosphoryl group of ATP to a nucleoside diphosphate. • NDP Kinase displays a steady state kinetic pattern that is distinctly different from that of adenylate kinase. If one substrate is varied while the other is fixed at several different concentrations, a family of parallel lines is obtained by Lineweaver-Burk analysis. This is reminiscent of a Ping-Pong reaction.
Economy in the Evolution of Binding Sites • Since adenylate kinase and nucleoside diphosphate kinase catalyze very similar reactions, why don’t they proceed by similar mechanisms? • NDP kinase catalyzes a symmetrical reaction, whereas adenylate kinase does not. For NDP kinase, the product of the ping (MgADP) is similar in structure to the substrate for the pong (MgGDP). The only difference involves the purine rings of each nucleotide. • By using a ping-pong reaction, the enzyme can use just one binding site for the phsphoryl transfer.
UDP-Glucose Pyrophosphorylase This is a special type of sequential mechanism in which MgUTP must bind firs, before glucose-1-phosphate. There is no degree of randomness. Ordered binding also implies ordered product release.
UDP-Glucose Pyrophosphorylase • Steady State kinetic equation is similar to adenylate kinase. Therefore Lineweaver-Burk plots cannot distinguish the two forms of sequential reactions. Must do product inhibition studies. • Stereochemistry indicates inversion; however, incubation of the enzyme with radiolabeled UTP, followed by gel-filtration shows a radiolabeled intermediate. Be careful! This is because UTP or UDP-glucose binds very tightly to the enzyme. In fact, the enzyme is isolated with UTP and UDP-glucose tightly bound, and will catalyze an exchange reaction, which is characteristic of Ping-pong reactions.
