Principles of Bioinorganic Chemistry - 2004 Note: The course seminar presentations will be held on Sunday, October 31, 2004 in the Bush Room. Please remember that daylight savings time ends that day.

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?

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.

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

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.

Enzymatic Catalysis of Urea Decomposition: Elimination or Hydrolysis? Guillermina Estiu and Kenneth M. Merz, Jr. pp 11832 - 11842

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.

Dioxygen Carriers: Hb, Mb, Hc, Hr Examples of Atom- and Group-Transfer Chemistry PRINCIPLES: Both substrate binding and redox changes occur Coupled proton-electron transfer steps set the redox potentials Closely positioned redox/acid-base units work in concert Interactions with substrates/other proteins gate electron transfer Two-electron transfer strategies include 2 metals, M-porphyrins Metal centers used to create or destroy radical species Changes in metal coordination spheres can facilitate allostery Bioinorganic chemistry of dioxygen paramount example ILLUSTRATIONS: O2 Binding and Transport: hemoglobin (Hb), myoglobin (Mb), hemocyanin (Hc), and hemerythrin (Hr) O2 Activation: cytochrome P-450, tyrosinase, methane monooxygenase; dioxygenases

Properties of Protein Dioxygen Carriers