Enzyme Properties Case Study I: Serine Protease Classification Active Sites Catalytic Strategies Protease I Convergence or Divergence Enzyme Inhibitions

Enzymes are Powerful and Highly Specific Catalysts The specificity of any enzyme is due to the precise interaction of the substrate with the enzyme. This precision is a result of the intricate three dimensional structure of the enzyme protein.

Classification of Enzymes by the Types of Reactions

Many Enzymes Require Cofactors for Activity Apoenzyme + cofactor = holoenzyme

Models of Enzyme-Substrate Binding Lock-any-key model The active site of the unbound enzyme is complementary in shape to the substrate. Induced-fit model The enzyme changes shape on substrate binding. The active site forms a shape complementary to the substrate only after the substrate has been bound.

Definitions  gated binding: binding that is controlled by the opening and closing of a physical obstacle to substrate or inhibitor access in the protein. reaction sub-site: that part of the active site where chemistry occurs.specificity sub-site: that part of the active site where recognition of the ligand takes place.

Specificity Nomenclature for Protease-Substrate Interactions The potential sites of interaction of the substrate with the enzyme are designated P (shown in red),and corresponding binding sites on the enzyme are designated S (subsite). The scissile bond (in red) is the reference point.

Common Features of The Active Sites of Enzymes The active site of any enzyme is the region that binds the substrates and the cofactors of any. It also contains the residues that directly participate in the making and breaking of bonds (catalytic groups). The active site is three-dimensional cleft formed by groups that come from different parts of amino acid sequence The active site takes up a relatively small part of the total volume of an enzyme. Active sites are clefts or crevices with good solvent accessibility. Substrates are bound to enzymes by multiple weak attractions. Active site of lysozyme.

Binding Sites for Small Ligands are Clefts, Pockets or Cavities In the active site of enzyme cytochrome P-450, the substrate (Camphor) is surrounded by residues from the enzyme. Heme is a cofactor and it completely buried inside the protein. There is no obvious route from the exterior to the active site pocket in the average structure.

Binding Sites for Macromolecules on a Protein’s surface can be Concave, Convex, or Flat The complex between human growth hormone and two molecules of its receptors Ribbon diagram of the complex. Two different protein-protein interfaces can be made by one molecule of growth hormone (yellow) with two identical receptor molecules (orange & green) A space-filling model of the complex shows the tight fit at both interfaces

Catalytic Sites Often Occur at Domain and Subunit Interfaces The two active sites are indicated by the presence of the bound cofactor NADPH (yellow). Each site occurs at an interface between the two subunits (blue and light-blue; red and brown) Structure of the dimeric bacterial enzyme 3-isopropylmalate dehydrogenase

Binding Sites generally Have a Higher than Average Amount of Exposed Hydrophobic Surface Surface view of the heme-binding pocket of cytochrome c6, with hydrophobic residues indicated in yellow The area around the heme (red) is very nonpolar because this protein must bind to another protein via this site to form an electron-transport complex involving the heme. The blue area indicates the presence o two positively charged residues important for heme binding.

Displacement of Water also Drives Binding Events Ligand binding involving hydrohhobic and hydrogen-bond interactions The binding of the lipid oleate (green) to the maize lipid-transport protein nsLTP involves mainly hydrophobic interactions of uncharged polar or nonpolar residues with the lipid tail, and hydrogen bonds (red dotted lines) to the charged head group. Affinity: the tightness of a protein-ligand complex Anisotropic: Behaving differently in different directions; dependent on geometry and direction Contribution to binding affinity can sometimes be distinguished from contributions to binding specificity.

Reactive Groups in Enzyme Active Sites are Optimally Positioned to Interact with the Substrate The enzyme uses a pyridoxal phosphate (PLP) cofactor (purple) and lysine (yellow outline) to carry out chemistry. The substrate amino acid (green) reacts with the cofactor to form an adduct (as shown in this model) which then rearranges to give product. Substrate specificity for the negatively charged aspartic acid substrate is determined by the positively charged guanidino groups of arginine 386 and arginine 292, which have no catalytic role. Mutation of arginine 292 to aspartic acid produces an enzyme that prefers arginine to aspartate as a substrate. Adapted from Cronin, C.N. and Kirsch, J.F.: Biochemistry 1988, 27:4572–4579; Almo, S.C. et al.: Prot. Eng. 1994, 7:405–412. the active site of E. coli aspartate aminotransferase

Protein Functions such as Molecular Recognition and Catalysis depend on Complementarity Substrate binding to anthrax toxin lethal factor Lethal factor (LF) a component of anthrax toxin acts as a protease to cut mitogen-activated protein kinase kinase (MAPKK-2), thereby blocking the cell cycle. This picture shows part of the surface of LF colored by charge. (Red: negative; Blue: positive) The model of the MAPKK-2 amino-terminal peptide shown in ball-and-stick representation. The active cleft of LF is complementary in shape and charge distribution to the substrate. Nature 2001, 414:229-233

Molecular Recognition Depends on Specialized Microenvironments that result from tertiary structure Specialized Microenvironments at Binding Sites Contribute to Catalysis Schematic of the active site of mandelate racemase showing substrate bound Lysine 164 is located very close to the catalytically important residue lysine 166. The proximity of these two positive charges lowers the proton affinity of both of them, making lysine 166 a better proton shuttle for the metal-bound substrate

Tight fit between a protein and its ligand The Flexibility of Protein Structure Allows Proteins to Adapt to their Ligand Tight fit between a protein and its ligand A space-filling representation of the catalytic domain of protein kinase A (blue) bound to a peptide analog (orange) of its natural substrate shows the snug fit between protein and ligand, achieved by mutual adjustments made by the two molecules.

Protein Flexibility is Essential for Biochemical Function HIV protease, an enzyme from the virus that causes AIDS, bound to three different inhibitors The anti-viral action of some drugs used in AIDS therapy is based on their ability to bind to the active site of viral protease and inhibit the enzyme. The protease inhibitors haloperidol (a) and crixivan (b) are shown, with a peptide analog (c) of the natural substrate also shown bound to the enzyme. Each inhibitor clearly ahs a quite different structure and two of them (a, b) are not peptides, yet all bind tightly to the active site and induce closure of a flap that covers it, a conformational change that also occurs with the natural substrate. (PDB 1aid, 1hsg, 1a8k)

The Degree of Flexibility Varies in Proteins with Different Functions The enzyme adenylate kinase can adopt either an open or closed conformation depending on which substrates are bound. In the presence of AMP alone, no conformational change occurs. On binding of the cosubstrate ATP, here in the form of the analog AMPPNP, a large rearrangement occurs that closes much of the active site. (PDB 2ak3, 1ank) Example of a large conformational change

The electrostatic potential around the enzyme Cu,Zn-superoxide dismutase Red contour lines indicate net negative electrostatic potential; blue lines net positive potential. The enzyme is shown as a homodimer (green ribbons) and two active sites (one in each subunit) can be seen at the top left and bottom right of the figure where a significant concentration of positive electrostatic potential is indicated by the blue contour lines curving away from the protein surface. The negative potential elsewhere on the protein will repel the negatively charged superoxide substrate (O2-·) and prevent non-productive binding, while the positive potential in the active site will attract it. Graphic kindly provided by Barry Honig and Emil Alexov.

Ways of Electrostatic Interactions can Influence the Binding of a Ligand to a Protein (a) Electrostatic forces and torques can steer the ligand (green) into its binding site on the protein (shown in yellow). (b) Some binding sites are normally shielded from the solvent and can be kept "closed" by salt links between groups on the protein surface. If the correct substrate disrupts these salt links it can gain access to the binding site. This is known as "gated" binding. Alternatively, the dynamics of the protein may open and close such a site transiently (as indicated by the yellow arrows). (c) Electrostatic interactions, particularly salt links and hydrogen bonds, between ligand and protein can contribute to the affinity and specificity of binding and to the orientation of the ligand in the binding site and the structure of the complex formed. All three of these ways of exploiting electrostatic interactions can be used by a single enzyme. Adapted from Wade, R.C. et al.: Proc. Natl Acad. Sci. USA 1998, 95:5942–5949.

Some Active Sites Chiefly Promote Proximity Catalysis of the reaction of carbamoyl phosphate and aspartate by the enzyme aspartate transcarbamoylase depends on holding the substrates in close proximity and correct orientation in the active site The reaction catalyzed by aspartate transcarbamoylasae (ATCase). Carbomoyl phosphate (pink) and aspartate (blue) undergo a condensation reaction to form N-carbamoyl aspartate. This is an essential step in pyrimidine biosynthesis. Schematic diagram of the ative site of APCase with the inhibitor PALA (green) bound. The amino acids forming the active sites and binding PALA by noncovalent bonds (red dotted lines) are represented by purple shapes. PALA resembles both carbamoyl phosphate and aspartate, as can be seen by comparison with (a), and binds to both the binding sites for these substrates in the active site.

Catalytic Strategies Covalent catalysis General acid-base catalysis The active site contains a reactive group, usually a powerful neucleophile that becomes temporarily covalently modified in the course of catalysis. General acid-base catalysis A molecule other than water plays the role of a proton donor or acceptor. Metal ion catalysis Metal ions can function catalytically 1) as an electrophilic catalyst to stabilize negatively charge on a reaction intermediate, 2) generate a nucleophile by increasing the acidity of a nearby molecules, and 3) binding to the substrate to increasing the interactions with the enzyme. Catalysis by approximation Reaction rate of two more distinct substrates can be enhanced by binding these substrates together along a single binding surface on an enzyme. Hydrolysis of chymotrypsin in two stages of acylation and deacylation (covalent hydrolysis).

Nucleophilic Catatlysis

The digestion of dietary protein begins in the stomach with the action of pepsin and continues in the small intestine with the action of trypsin, chymotrypsin and elastase along with a series of exopeptidases and dipeptidases, some derived from the pancreas and some from the intestinal mucosal cells.

The activation of trypsin by enteropeptidase (enterokinase) triggers the activation, in turn, of other endopeptidases as well as the exopeptidases, carboxypeptidases A and B.

Degradation of Peptide Amino peptidase Dipeptidase These enzymes are metalloproteases and typically require the metals Mn2+ or Zn2+ for action The net result of the action of pancreatic, gastric, and small intestine proteases is the degradation of dietary proteins to amino acids, dipeptides, and tripeptides all of which can be absorbed By the digestive system.

Major Classes of Proteases Serine proteases Cysteine protease Aspartyl Proteases Metalloproteases

Major Classes of Proteases

Serine Proteases Serine proteases possess a highly reactive serine residue in addition to His and Asp to form a catalytic triad. The major mammalian pancreatic enzymes –trypsin, chymotrypsin, and elastase are kinetically very similar. They catalyze and hydroplyzepeptides and synthetic ester substrates

Peptide Hydrolysis by Chymotrypsin Ser-195 makes a nucleophilic attack on the carbonyl carbon atom of the substrate to form a tetrahedral intermediate of oxyanion hole (covalent catalysis). Later the water molecule attacks the carbonyl group while a proton is concomitantly removed by the His residue (general acid catalyst) forming a tetrahedral intermediate (step 6).

Serine Protease -Chymotrypsin Chymotrypsin has a specificity to cleave proteins on the carboxyl side of aromatic or large hydrophobic amino acids.

Specificity of Trypsin vs. Thrombin (A) Trypsin cleaves on the carbonyl side of Arg, whereas (B) thrombin cleaves Arg-Gly bonds in particular sequences specifically The specificity of any enzyme is due to the precise interaction of the substrate with the enzyme. This precision is a result of the intricate three dimensional structure of the enzyme protein.

Specificity is Determined by the Substrate Binding Pocket Thr-226 Gly-216, Gly-216)

Comparison of the Binding Pockets (left) Chymotrypsin with N-formyl-L-Trp bound and (right) elastase with N-formyl-L-Ala bound. Trypsin has one mutation of S189D at the active site.

Divergent Evolution of Protein Families The structures and sequences of mammalian serine proteinases are very similar. Chymotrypsin (red) and trypsin (blue) has > 50% sequence similarity, suggesting that they are presumably derived from a common ancestor (divergent evolution)

Divergent Evolution can Produce Proteins With Sequence and Structural Similarity but Different Functions Ribbon diagram of the structure of a monomer of benzoylformate decarboxylase (BFD) and pyruvate decarboxylase (PDC). BFD (top) and PDC (BOTTOM) shares a common fold and overall biochemical function, but they recognize different substrates and have low (2%) sequence identity. The bound thiamine pyophosphate cofactor is shown in space-filling representation in both structures. The green spheres are metal ions. (PDB 1bfd and 1pvd) Superposition of the three-dimensional structures of steroid-delta-isomerase, nuclear transport factor-2, and scytalone dehydratase. The active site is indicated by an arrow. (PDB 8cho, 1oun, 1std)

Convergent Evolution Different organisms, starting from different structures have evolved a common mechanism. The bacterial protease subtilisin from Bacillus amyloliquefaciens has no sequence and structure homology to mammalian serine protease. It has the same catalytic triad The structure of carboxypeptidase II from wheat has two chain without structural similarity to chymotrypsin. It also has a catalytic triad.

The Structure Folds of Chymotrypsin vs Substilisin The overall folds of two members of different superfamilies of serine protease. The enzymes are chymotrypsin (top) and subtilisin (bottom). The residues in the catalytic triad are indicated for each)

Convergence or Divergence? Six Criteria for testing whether two proteins have evolved from a common precursor: The DNA sequences of their genes are similar Their amino acid sequences are similar Their three-dimensional structures are similar Their enzyme-substrate interactions are similar Their catalytic mechanisms are similar The segments of polypeptide chain essential for catalysis are in the same sequence (i.e. not transposed). These criteria are in descending order of strength. Sometimes, the structure has been conserved through evolution but function has been changed.

Protein Family and Superfamily Family: a group of homologous proteins that share a related function. Members of the same enzyme family catalyze the same chemical reaction on structurally similar substrates. Superfamily: protein with the same overall fold but with usually less than 40% sequence identity. The nature of the biochemical functions performed by proteins in the same superfamily are more divergent than those within families. A comparison of primer-template DNA bound to three DNA polymerases. (a) Taq DNA polymerase bound to DNA. The DNA stacks against the “fingers” and is contacted across the minor groove by the “thumb” domain. (b) The binary complex of HIV-1 reverse transcriptase and DNA. This structure does not have a nucleotide-binding alpha helix in the gingers domain. Instead, a beta hairpin probably performs this function. (c) The ternary complex of rat DNA polymerase beta with DNA and deoxy-ATP (not shown). Although this polymerase has an additional domain (A), the “thumb” domain similarly binds the DNA primer-template in the minor groove, while the “fingers” present a nucleotide-binding alpha helix at the primer terminus. (PDB 1tau, 2hmi, 8icp)

Enzyme Inhibitions Irreversible inhibitions-inhibitors dissociates very slowly from its target enzyme because if it has become tightly bound to the enzyme either covalently or noncovalently. Reversible inhibition-rapid dissociation of the enzyme-inhibitor complex. A competitive inhibitor binds at the active site and prevent the substrate from binding A noncompetitive inhibitor does not prevent the substrate from binding.

Enzyme Inhibitions by Irreversible Inhibitors By group specific reagents react with specific R group of amino acids DIPF for Ser and iodoacetamide for Cys By substrate analogs-affinity labels that covalently modify active site residues By suicide inhibitors

Affinity Labeling Tosyl-L-phenyalanie chloromethyl ketone (TPCK) is a reactive analog of the normal substrate for the enzyme chymotrypsin. TPCK binds at the active site of chymotrypsin and modifies an essential His residue. Bromoacetol phosphate, an analog of dihyoxyacetone phosphate, binds at the active site of triose phosphate isomerase (TIM)and covalently modifies a Glu residue required for the enzyme activity.

Mechanism-based (Suicide) Inhibition The suicide inhibitor binds to the enzyme as a substrate and is initially processed by the normal catalytic mechanism. A chemically reactive intermediate is generated and in turn inactivate the enzyme through covalent modification.

Case Study III Zinc Metalloenzymes September 27, 2004 CPA 1.5 Ǻ Pdb:1m4l Pdb:1dtd CPA+ inhibitor 1.65 Ǻ September 27, 2004

Properties of Zinc The abundance of zinc in biology is second only to that of iron among the transition and group II elements The zinc ion contains a filled d orbital (d10) and therefore does not participate in redox reactions but rather functions as a Lewis acid to accept a pair of electrons Colorless, spectroscopically silent Electronic configuration

A Growing Awareness of Zinc in Biology The first zinc metalloenzyme, carbonic anhydrase II (EC 4.2.1.1), was discovered in 1940 by Keilin and Mann >300 zinc enzymes covering all six classes of enzymes have thus far been discovered in different species pdb:2cbd Human CA II

For histidine, which N atom is more preferable for binding? Zinc Binding Ligands Zn2+ can coordinate with atoms N, O or S In protein zinc-binding sites, Zn2+ is coordinated by different combinations of protein side chains, including the nitrogen of histidine (H), the sulfur of cysteine (C), the oxygen of aspartate (D) and glutamate (E) H C Other more rarely observed ligands include the hydroxyl of tyrosine (Y), the carbonyl oxygen of the protein backbone and the carbonyl oxygen of either glutamine (Q) or asparagine (N) D E ?? For histidine, which N atom is more preferable for binding? Nε or Nδ ?

Zinc Binding Ligands The majority of histidine zinc ligands found in zinc protein structures coordinate zinc through the Nε atom. The zinc ion prefers a head-on and in-plane approach to the sp2 lone pair of the N atoms.

Zinc Binding Geometry Zinc can have coordination of numbers from 2 to 8 in zinc complexes, 4, 5, and 6 are most frequently found in biological systems T4 T5 T6

Zinc Binding Geometry Zn (II) has a ligand-field stabilization energy of zero in all liganding geometries, and hence no geometry is inherently more stable than another Its stereochemical flexibility likely contributes to catalysis because it can transiently accept different coordination geometries without impeding catalysis When cysteine-specific tRNA synthetase binds its cysteine substrate, the coordination of the protein-bound zinc ion changes from tetrahedral (left) to trigonal-bipyramidal (right) to create an extra binding site(© K. J. Newberry et al, EMBO J., 2002, 21, 2778)

Zinc Binding Sites Catalytic sites Cocatalytic sites Structural sites Protein interface sites Protein interface Zinc binding sites in enzymes: catalytic (thermolysin), structural (alcohol dehydrogenase), cocatalytic (aeromonas proteolytica aminopeptidase) Protein interface sites: zinc may also influence the quaternary structure of proteins. (Left): gamma-carbonic anhydrase; (right) superantigen, staphyloccus enterotoxin C2.

Comparison of Zinc Binding Sites Comparison of the zinc ligands (L1, L2 and L3) and the spacers (X, Y and Z) between zinc ligands in catalytic and structural zinc sites

Comparison of Zinc Binding Sites Frequency of protein residues acting as zinc ligands in catalytic and structural zinc sites

Zinc Metalloprotease Family Families of zinc metalloproteases based on the sequence around the zinc-binding residues

Unique Feature of Zinc Enzymes The unique feature of most zinc enzymes is the presence of an activated WATER molecule bound to Zn(II). The pKa of metal-free water is 15.7 but can be reduced to 10 in [Zn(H2O)6]2+ and 7 with three N-donors. This allows for facile ionization of H2O carbonic anhydrase II alcohol dehydrogenase carboxypeptidase A

Unique Feature of Zinc Enzymes The zinc-bound water is a critical component for a catalytic zinc site: ionized to zinc-bound hydroxide (as in CA) polarized by a general base to generate a nucleophile for catalysis (as in Carboxypeptidase A) displaced by the substrate (as in alkaline phosphatase)

Unique Feature of Zinc Enzymes In the zinc proteases, the zinc ion serves as a powerful electrophilic catalyst by providing all or a combination of the following: An activated water molecule for nucleophilic attack Polarization of the carbonyl of the scissile bond Stabilization of the negative charge in the transition state

Reactions Catalyzed R X H O A + c o n d e s a t i h y r l . g = N ' p 2 c o n d e s a t i h y r l . g = N ' p , m P 3 -

Carboxypeptidase A: An Example of Catalysis by zinc H196 H69

Carboxypeptidase A A pancreatic enzyme that cleaves the carboxyl terminal amino acid from a peptide chain by hydrolyzing the amide linkage. High selectivity for substrates with large terminal aliphatic or phenyl substituents …Pro-Leu-Glu-Phe ...Pro-Leu-Glu + Phe H2O carboxypeptidase A

Zinc ligands of carboxypeptidases (CPDs) Carboxypeptidase A Zinc ligands of carboxypeptidases (CPDs)

X-ray Structures of Active Sites Carboxypeptidase A Carboxypeptidase G2

Carboxypeptidase A How CPA works? Active site Physical properties 307 amino acid residues plus one Zn2+ ion MW: 34,600 Da Roughly egg-shaped with approx. dimensions 50 Å x 38 Å Active site Histidine-196 Histidine-69 Glutamate-72 A cleft on one side contains the Zn2+ ion = active site The zinc ion is coordinated by two N atoms (histidines) and two O atoms (glutamate) in the protein chain; the fifth site is H2O. ?? How CPA works?

Dipolar interaction H-bonding H-bonding

Nucleophilic attack on carbonyl C-atom Ionization

Cleavage of the C-N bond

Proton transfer to give NH3+ & carboxylate

Thermolysin: Enhancing Thermostability by Engineering

Thermolysin A thermostable neutral metalloproteinase isolated from Bacillus thermoproteolyticus Requires essentially one zinc ion for enzyme activity and four calcium for structural stability Scanning Electron Micrograph of Bacillus Bacteria http://www.biosci.ohiou.edu/introbioslab/Bios170/170_5/bacillus.html

Zinc ligands of thermolysins (TLs).

Engineering TL-like Protease from Bacillus Stearothermophilus Mutant with hyperthermal stability Red: mutated sites Yellow: -s-s- crossing-linking residues The engineered protease exhibits a 21 ℃ increase in the temperature optimum for activity (PNAS 1998, 95, 2056-60)

Enhanced Thermostability by incorporation of Aib H3C Thermal unfolding of the natural and engineered C-terminal residues 255-316 of thermolysin Ala304Aib/Ala209Aib Wild type Ala→Aib (α-aminoisobutyric acid) a natural, non-protein amino acid found in some microbial species. exclusively forms and stabilizes helical backbone structure. possesses a higher intrinsic helical propensity than Ala, which is the most helix-favoring among the 20 amino acids. (Biochemistry 1998, 37, 1686-96)