Principles of Bioinorganic Chemistry - 2003 You should have your paper topic approved by Prof. Lippard this week, if you have not done so already (by 10/12 please). The oral presentations will be held in research conference style at MIT's Endicott House estate in Dedham, MA, on Saturday, October 18. WEB SITE: web.mit.edu/5.062/www/

Hydrolytic Enzymes, Zinc and other Metal Ions PRINCIPLES: M(OH)n+ centers supply OH- at pH 7 by lowering water pKa Mn+ serves as general Lewis acid, activating substrates Rate acceleration occurs by internal attack within coord. sphere Protein side chains greatly assist assembly of transition state Carboxylate shifts can occur, especially at dimetallic centers Electrostatic interactions predominate Non-redox active metal ions often but not universally used Illustrating the Principles: Carboxypeptidase, carbonic anhydrase - delivering hydroxide Alcohol dehydrogenase: an oxidoreductase Dimetallic metallohydrolases: are two metals better than one?

Carboxypeptidase A: A Hydrolytic Zinc Enzyme Reaction catalyzed: R–CH–C(O)–NH–R’ R–CH–CO2- + +NH3–R’ NH2R’’ NH2R’’ Cleaves C-terminal peptide bonds; prefers aromatic residues. Active site contains a single catalytic zinc, essential for activity. The glutamate can undergo a carboxylate shift. Thermolysin has a similar active site; it is an endopeptidase.

Carboxypeptidase A structure with the inhibitor glycyl-L-tyrosine bound at the active site. Note hydrogen bonds to key residues in the active site that position the substrate moiety for bond scission.

Catalytic Mechanism for Carboxypeptidase A Summary of events: 1. Substrate binds; orients by the terminal carboxylate. 2. Deprotonate bound H2O. 3. Polarize scissile bond by Arg127. 4. Bound OH- attacks peptide C(O). 5. Form tetrahedral transition state. 6.Lose 2 peptide fragments and recycle the enzyme. Principles illustrated: 1. Zinc serves as template. 2.Metal supplies cleaving reagent, OH-, and organizes key groups. 3. Chemistry achieved at neutral pH! Kcat ~ 100 s-1 .

Carbonic Anhydrase, the First Known Zn Enzyme Reaction catalyzed: CO2 + H2O H2CO3 ~ 106 s-1

Carbonic Anhydrase PZn(OH2)2+ PZn(OH)+ + H+ Keq = 10-7 M = kf/kr Note: Rate 10-2 s-1 at pH 7; kf 106 s-1 in active site. Paradox: The reverse reaction is diffusion controlled, with kr ~ 1011 M-1 s-1 Thus kf ≤ 104 s-1. So how can the turnover be 106 s-1 ? Answer: Facilitated diffusion of protons by buffer components bound to the enzyme.

Possible Carbonic Anhydrase Mechanism

Alcohol Dehydrogenase, an Oxidoreductase Reaction catalyzed: RCH2 OH + NAD+ RCHO + NADH + H+ Enzyme contains two 40 kDa polypeptides, each with 2 Zn2+centers in separate domains. One zinc is structural, the other catalytic. Catalytic zinc is 20 Å from the surface, near the nicotinamide binding region. This center is not required for NAD + cofactor binding. Alcohol substate DO require zinc and bind directly to the metal center, displacing the coordinated water.

Schematic Diagram NAD+ binding to the active site of LADH, with specific, well-positioned amino acid side chains holding it in place. Ethanol is shown bound to the zinc, displacing water. The system is set to undergo catalysis.

Note hydride transfers from a-C of alcohol to nicotinamide ring.

Alkaline Phosphatase; a Dizinc(II) Center Activates the Substrate 1. The substrate binds to the dizinc center; a nearby Arg also helps activate it. 2. A serine hydroxyl group attack the phosphoryl group, cleaving the ester. The phosphate is transferred to the enzyme, forming a phosphoryl-serine residue. 3. Hydrolysis of this phosphate ester by a zinc-bound hydroxide com-pletes the catalytic cycle. This mechanism is supported by studies with chiral phosphate esters (ROP18O17O16O)2-; there is no net change in chirality at phoshorus. 1. 3. 2.

Dimetallics can move the value into the physiological range near pH 7

Advantages of Carboxylate-Bridged Dimetallic Centers in Chemistry and Biology

Principles illustrated: the dimetallic affords hydroxide; the substrate is positioned by residues in the active site; the dimetallic stabilizes the urea leaving group; redox inactive metal; electrostatics

Metallo-b-lactamases, an Emerging Clinical Problem PZn(OH2)2+ PZn(OH)+ + H+ Keq = 10-7M = kf/kr

b-Lactamase from Bacteroides fragilis Zn2 Wat2 C181 Wat1 Zn1 H99 H101 H162 D103 H223 N.O. Concha, B.A. Rasmussen, K. Bush, O. Herzberg (1996), Structure 4, 823-836

Summary - Points to Remember Both mono- and dimetallic centers lower the pKa value of bound water, allowing hydroxide to be delivered at pH 7. Coordination of the leaving group portion of the substrate to a metal ion activates the substrate for nucleophilic attack. Residues not coordinated but in the second coordination sphere can participate directly (serine in phophatases) or indirectly (arginine in alcohol dehydrogenase) in substrate attack, orientation, and/or activation. Carboxylate shifts facilitate substrate binding, activation. Redox inactive metal ions (Zn2+, Ni2+, Mn 2+, Co2+) preferred.