ChemLecture12

The increase in ATCase activity in response to increased ATP concentration has two potential physiological explanations:

1. High ATP concentration signals a high concentration of purine nucleotides in the cell -the increase in ATCase activity will tend to balance the purine and pyrimidine pools 2. A high concentration of ATP indicates that there is significant energy stored in the cell to promote mRNA synthesis and DNA replication and leads to the synthesis of pyrimidines

Mg2+ has three essential functions:

1. Holds the nucleotide in well defined conformations that can be specifically bound to the enzyme 2. Neutralize polyphosphate charges, reducing nonspecific ionic interactions 3. Provide additional points of interaction with the enzyme, either directly to the Mg2+ ions, or indirectly with the Mg2+ ions through H bonds to the coordinated H2O molecules

Mechanism of Carbonic Anhydrase

1. Zinc ion allows the generation of a hydroxide ion 2. CO2 binds to the enzyme and is positioned to react with the hydroxide ion 3. Hydroxide ion attacks the CO2 and converts it into bicarbonate ion 4. The active site is regenerated with the release of bicarbonate and the binding of another water molecule

ATCase has Separable Catalytic and Regulatory Subunits

ATCase can be separated into regulatory (r) and catalytic (c) subunits with the addition of p- hydroxymercuribenzoate p-hydroxymercuribenzoate reacts with cysteine residues This was followed by ultracentrifugation to separate the subunits Subunits differ in size which allows separation Native enzyme - 11.6S Dissociated subunits - 2.8S and 5.8S The larger subunit is the catalytic subunit Has catalytic activity but does not respond to CTP and does not exhibit sigmoidal behavior Catalytic subunit has three chains (34 kDa each) (trimer) The smaller regulatory subunit can bind CTP but has no catalytic activity Regulatory subunit has two chains (17 kDa each) (dimer) These subunits combine rapidly when mixed to yield two catalytic trimers and three regulatory dimers in the full complex

How is this enzyme regulated to generate precisely the amount of CTP needed by the cell?

ATCase is inhibited by CTP, the final product of the ATCase controlled pathway Effect of CTP on the enzyme exemplifies negative feedback inhibition Feedback inhibition prevents the needless accumulation of product These are called allosteric sites

Conformational Changes in Adenylate Kinase

ATP-Mg2+ binding causes conformational change P-loop closes down on polyphosphate chain, especially, β phosphoryl group, top domain moves down to form lid over bound nucleotide. Thus, its γ phosphoryl group is positioned next to the binding site for 2nd substrate, NMP. Binding of NMP induces additional conformational changes. Catalytically competent conformation now formed, preventing transfer of phosphoryl group to water. Thus, enzyme stabilizes the transition state that leads to the transfer of the phosphoryl group from ATP to NMP

Aspartate Transcarbamylase (ATCase)

Aspartate transcarbamoylase catalyzes the first step in the biosynthesis of pyrimidines, bases that are components of nucleic acids. Condensation of aspartate and carbamoyl phosphate to form N-carbamoylaspartate and orthophosphate products react many times to produce CTP.

Restriction Enzymes

Bacteria have evolved restriction enzymes (endonucleases) to protect themselves from foreign viral DNA Restriction enzymes degrade the viral DNA upon entry into the cell Restriction enzymes must be highly specific to avoid damaging host EcoRV cleaves foreign DNA containing the sequence GATATC but leaves host genome intact even though it also contains this sequence. Why? Restriction enzymes will not cleave DNA if it is methylated

catalysis by approximation

Both sets of changes only occur when both the donor and acceptor are bound thus preventing transfer of the phosphoryl group to water Bringing the substrates into proximity at the proper orientation is an example of catalysis by approximation

Proton Shuttle

Buffer molecules too large to access active site, therefore evolve proton shuttle Allows buffer components to participate in the reaction from solution Histidine residue transfers protons from the zinc-bound water molecule to the protein surface and then to the buffer Catalytic function enhanced with proton shuttle that transfers protons to and from the active site

Buffer Participation

Buffer participates in the reaction If buffer component BH+ has a pKa of 7 (same as Zn bound H2O), then, K for the reaction is = 1 Rate of proton abstraction given by k1 x [B] Rate constants k1 & k-1 limited by buffer diffusion values < 109 M-1s-1 Buffer concentrations [B] > 1mM enough to give hydration rate of 106 M-1s-1, because, k1 x [B] = (109 M-1s-1) x (10-3 M) = 106 s-1 The rate of carbon dioxide hydration increases directly with the concentration of buffer The buffer allows carbonic anhydrase to achieve its high catalytic rates

Effect of CTP on ATCase

CTP inhibits action of ATCase Structural studies revealed that the enzyme is in the T state when bound to CTP binding site for CTP in each regulatory chain in a domain that does not interact with the catalytic subunit Binding of the inhibitor CTP shifts the equilibrium toward the T state, decreasing the net enzyme activity

Kinetic curve differs from that expected for an enzyme that follows Michaelis-Menten kinetics

Called sigmoid because it resembles an "S" Sigmoidal curves result from cooperation between subunits Coooperativity: The binding of substrate at one active sites increases the activity at the other active site

Kinetics of Water Deprotonation

Carbon dioxide is hydrated at a rate of 106/s, however rate of proton diffusion limits rate of H+ release to < 104/s for a group with pKa = 7 Water must be deprotonated first before catalysis can occur Why is the rate of CO2 hydration greater than the rate of water deprotonation?

Structures of two isomeric forms of ATP-Mg2+

Could for with two oxygens alpha and gamma beta and gamma Only oxygen coordination shown Magnesium ions are coordinated to six groups (2 oxygen, 4 waters) in an octahedral arrangement Usually two oxygen atoms are directly coordinated to the magnesium ion with other positions occupied by water molecules (4 waters)

EcoRV Restriction Enzymes - Specificity

EcoRV has two protein subunits (yellow and blue) which bind to the DNA substrate (red) The two-fold axes of both the enzyme dimer and the DNA are aligned

Equilibrium Between R and T States

Even in the absence of any substrate or regulators, ATCase exists in equilibrium between the R and T states The T state is favored under these conditions by a factor of 200

The distortion of DNA explains how methylation blocks cleavage to protect host DNA

Host methylates at the amino group of the adenine nucleotide at the 5' end of the recognition sequence The presence of a methyl group prevents the formation of a hydrogen bond between the amino group and the side-chain carbonyl of Asn 185 The absence of this hydrogen bond disrupts other interactions between the enzyme and the DNA Therefore, no distortion of the DNA takes place

Basis for Allostery

How can we explain the enzyme's sigmoidal kinetics? In the absence of substrate, the enzyme exists almost entirely in the T state Binding of substrate molecules to one active site increases the likelihood of the entire enzyme shifting towards the R state The presence of more substrate will increase the fraction of enzyme in the more active R state → called cooperativity ATCase largely follows the concerted mechanism because the change in the enzyme is "all or none"

ATP is also an allosteric regulator of ATCase

However, the effect of ATP is to increase the reaction rate at a given aspartate concentration At high concentrations of ATP, the kinetic profile shows a less pronounced sigmoidal behavior

CTP increases the initial phase of the sigmoidal curve

In the presence of CTP, the enzyme becomes less responsive to the cooperative effects facilitated by substrate binding Therefore, more substrate is required to attain a given reaction rate

ATP-Mg2+ complex bound to adenylate kinase

Magnesium ion is bound to the phosphoryl groups as well as to water molecules The water molecules interact with groups on the enzyme including a conserved aspartate residue The magnesium ion provides additional points of interaction between the ATP-Mg2+ complex and the enzyme

P-Loop

NMP kinases form a family of homologous proteins Conserved NTP binding domain present consisting of a central beta sheet surrounded on both sides with alpha helices Important feature is a loop between the beta sheet and the first alpha helix Referred to as P-loop because it interacts with the phosphate groups on the NTP P-loops found in other important nucleotide-binding proteins

Structural isomers of ATP-Mg2+

NMP kinases require magnesium or manganese ions for activity However, these ions are not part of the active site NTPs, such as ATP, bind to these metal ions and this ATP-Mg2+ complex is the true substrate for the enzyme Interactions between the magnesium ions and the phosphoryl groups holds the nucleotide in a well-defined conformation that can be recognized by the enzyme

NMP Kinases

Nucleoside monophosphate (NMP) kinases catalyze transfer of terminal phophate from an NTP (usually ATP) to the phosphate group on a nucleoside monophosphate (NMP) The challenge for NMP kinase is to promote this transfer without promoting the competing reaction of transferring the phosphate group from ATP to water (NTP hydrolysis)

Allosteric Effects

Observation that ATCase is inhibited by CTP is remarkable because CTP is structurally quite different from the substrates of the reaction Therefore, CTP must bind to a site distinct from the active site where substrate binds CTP is an example of an allosteric inhibitor In ATCase (but not all allosterically regulated enzymes), the catalytic sites and the regulatory sites are on separate polypeptide chains Allosterically regulated enzymes do not follow Michaelis-Menten kinetics

P-loop Interaction with ATP

P-loop highly conserved The P-loop of adenylate kinase interacts with the phosphoryl groups of ATP Hydrogen bonds (green) link ATP to peptide NH groups and a lysine residue, which is highly conserved among NMP kinases

PALA - Bisubstrate Analog

PALA , a bisubstrate analog (an analog of the two substrates) that resembles an intermediate along the pathway of catalysis PALA is a potent competitive inhibitor of ATCase; it binds to and blocks the active sites Enzyme was crystallized in the presence of PALA to locate the active sites

Basis for Allostery

R & T states equivalent to 2 enzymes with different Kms The R state curve has a low Km and the T state curve has a very high Km Sigmoid curve can be pictured as a composite of two Michaelis- Menten curves, one corresponding to the T state and the other to the R state An increase in substrate concentration favors a transition from the T-state curve to the R state curve enzymes with different Kms

Restriction Enzymes - Specificity

Recognition sequences for most restriction enzymes are inverted repeats The 3D structure has two-fold rotational symmetry This requires that the enzyme have similar symmetry

Restriction Enzymes - Mechanism

Restriction enzymes catalyze the hydrolysis of the phosphodiester backbone of DNA The bond between the 3'-oxygen atom and the phosphorus atom is broken The products are a free 3'-hydroxyl group and a 5'-phosphoryl group at the cleavage site Type II restriction enzymes best characterized Type II enzymes cut within the recognition sequence

Regulatory Strategies

The activity of enzymes has to be regulated, so that they function at the proper time and place There are four basic regulatory strategies: 1. Allosteric control. Proteins contain distinct regulatory sites and multiple functional sites. Binding of regulatory molecules triggers conformational changes that affect the active sites. Display cooperativity: small [S] changes - major activity changes Information transducers: signal changes activity or information shared by sites 2. Multiple forms of enzymes (isozymes). Used at distinct locations or times. Differ slightly in structure, in Km & Vmax values, and in regulatory properties. Still catalyze the same reactions 3. Reversible covalent modification. Activities altered by covalent attachment of modifying group, mostly a phosphoryl group 4. Proteolytic activation. Irreversible conversion of an inactive form (zymogen) to an active enzyme

Zinc Activation of a Water Molecule

The effect of pH on carbonic anhydrase activity was the first clue that zinc was involved in the reaction Transition near pH 7.0 Suggests a group with pKa = 7 is important Group is zinc - bound water molecule The binding of a water molecule to the positively charged zinc reduces the pKa of the water molecule from 15.7 to 7 With a much lower pKa, many water molecules lose a proton at pH 7 which generates a significant amount of hydroxide ion bound to the zinc ion Zinc-bound hydroxide ion is a very potent nucleophile that can more readily attack carbon dioxide

Regulatory Strategies - Model Systems

The following proteins are used as model systems exemplifying each type of regulatory mechanism: 1. Aspartate transcarbamoylase is allosterically inhibited by the end product of its pathway 2. Hemoglobin transports O2 efficiently by binding it cooperatively 3. Isozymes provide a means of regulation specific to distinct tissues and developmental strategies 4. Phosphorylation by kinases as a means of regulating enzyme activity 5. Chymotrypsin activated by specific proteolytic cleavage

Kinking of the Recognition Site

The path of the DNA helical axis is substantially distorted on binding to the enzyme This facilitates breakage of the phosphate bond

DNA Distortion

The structures of complexes formed with noncognate DNA fragments are very different from that of the recognition sequence Noncognate DNA is not substantially distorted This lack of distortion has negative effects upon catalysis No phosphate group is positioned to complete the binding site for the magnesium ions Magnesium ions are essential for catalytic activity

Human Carbonic Anhydrase II

There are at least seven different carbonic anhydrases in humans Human carbonic anhydrase II is a major protein component and is well studied First known zinc containing enzyme - discovered in 1932 More than one third of all enzymes require metal ions Metal ions utilize the following properties to assist catalysis: 1. Positive charge 2. Can form relatively strong but kinetically labile bonds 3. In some cases, have the capacity to be stable in more than one oxidation state

What accounts for the exquisite specificity of EcoRV?

There are two loops within the enzyme (one from each subunit)that directly hydrogen bond to specific base pairs The DNA is distorted with a substantial kink The central TA sequence of GATATC does not bind to the enzyme This TA sequence is easily kinked Therefore, the enzyme can "read" the DNA sequence using hydrogen bonding with amino acids

ATCase Structure

Two catalytic trimers are stacked one on top of the other, linked by three dimers of the regulatory chains There are significant contacts between the two catalytic trimers Each r chain within a regulatory dimer interacts with a c chain within a catalytic trimer through a structural domain stabilized by a zinc ion bound to four cysteine residues p-hydroxymercuribenzoate is able to dissociate the catalytic and regulatory subunits because mercury binds strongly to the cysteine residues, displacing the zinc and destabilizing this domain

Structural Changes Associated with Allostery

Two catalytic trimers move 12 Å farther apart and rotate approximately 10 degrees about their common threefold axis of symmetry Regulatory dimers rotate approximately 15 degrees to accommodate this motion ATCase-PALA complex reveals a remarkable change in quaternary structure on binding of PALA enzyme literally expands on PALA binding Presence or absence of substrate or substrate analogs determines the state ATCase has two distinct quaternary forms: the T (for tense) state and the R (for relaxed) state T state has lower affinity for substrates and, hence, lower catalytic activity than does the R state The enzyme exists in equilibrium between the T and the R forms

Conserved Structure

Type II restriction enzymes have a conserved structure Single monomer shown for each enzyme Colored regions indicate conserved structural elements Eg. EcoRV, EcoRI, BamHI

Human Carbonic Anhydrase II and Zinc Site

Zinc ion is bound to the imidazole rings of three histidines as well as a water molecule Zinc site is located in a cleft near the center of the enzyme Zinc is always in the +2 state in biological systems The protonation state of the water molecule is pH- dependent

Carbonic Anhydrase

metal ion catalysis Carbon dioxide is a major end product of aerobic metabolism In mammals, carbon dioxide released into blood and transported to lungs KNOW RXN Carbon dioxide reacts with water inside red blood cells Even without enzyme, this reaction proceeds with a rate constant of 0.15/s However, reaction needs to be even faster for physiological processes Carbonic anhydrases greatly increase the rate of reaction Mutations in some carbonic anhydrases results in osteopetrosis (formation of dense bone and anemia) and mental retardation Human carbonic anhydrase II hydrates CO2 at a million times a second, (kcat = 106 s-1) Found in red blood cells CO2 hydration & HCO3 - dehydration coupled to rapid transport processes (tissue to blood, blood to lungs)

ATCase Active Sites

structure of the ATCase- PALA complex reveals that PALA binds at sites lying at the boundaries between pairs of c chains within a catalytic trimer Most of the residues belong to one subunit, but several key residues belong to a neighboring subunit Because the active sites are at the subunit interface, each catalytic trimer contributes three active sites to the complete enzyme