Reactivity of μ-Hydroxodizinc(II) Centers in Enzymatic Catalysis through Model Studies

Abstract

The stable dinuclear complex [Zn 2 (BPAN)(µ-OH)(µ-O 2 PPh 2 )](ClO 4 ) 2 , where BPAN ) 2,7-bis[2-(2-pyridylethyl)-aminomethyl]-1,8-naphthyridine, was chosen as a model to investigate the reactivity of (µ-hydroxo)dizinc(II) centers in metallohydrolases. Two reactions, the hydrolysis of phosphodiesters and the hydrolysis of -lactams, were studied. These two processes are catalyzed in vivo by zinc(II)-containing enzymes: P1 nucleases and -lactamases, respectively. The former catalyzes the hydrolysis of single-stranded DNA and RNA. -Lactamases, expressed in many types of pathogenic bacteria, are responsible for the hydrolytic degradation of -lactam antibiotic drugs. In the first step of phosphodiester hydrolysis promoted by the dinuclear model complex, the substrate replaces the bridging diphenylphosphinate. The bridging hydroxide serves as a general base to deprotonate water, which acts as a nucleophile in the ensuing hydrolysis. The dinuclear model complex is only 1.8 times more reactive in hydrolyzing phosphodiesters than a mononuclear analogue, Zn(bpta)(OTf) 2 , where bpta ) N,N-bis-(2-pyridylmethyl)-tert-butylamine. Hydrolysis of nitrocefin, a -lactam antibiotic analogue, catalyzed by [Zn 2 (BPAN)(µ-OH)(µ-O 2 PPh 2 )](ClO 4 ) 2 involves monodentate coordination of the substrate via its carboxylate group, followed by nucleophilic attack of the zinc(II)-bound terminal hydroxide at the -lactam carbonyl carbon atom. Collapse of the tetrahedral intermediate results in product formation. Mononuclear complexes Zn(cyclen)-(NO 3 ) 2 and Zn(bpta)(NO 3 ) 2 , where cyclen ) 1,4,7,10-tetraazacyclododecane, are as reactive in the -lactam hydrolysis as the dinuclear complex. Kinetic and mechanistic studies of the phosphodiester and -lactam hydrolyses indicate that the bridging hydroxide in [Zn 2 (BPAN)(µ-OH)(µ-O 2 PPh 2 )](ClO 4 ) 2 is not very reactive, despite its low pK a value. This low reactivity presumably arises from the two factors. First, the bridging hydroxide and coordinated substrate in [Zn 2 (BPAN)(µ-OH)(substrate)] 2+ are not aligned properly to favor nucleophilic attack. Second, the nucleophilicity of the bridging hydroxide is diminished because it is simultaneously bound to the two zinc(II) ions. IntroductionMetallohydrolases are hydrolytic enzymes that depend on metal ions, usually divalent zinc, cobalt, and manganese, for catalysis. 1 The mechanisms of enzymes containing a single metal ion, such as carboxypeptidase and carbonic anhydrase, have been extensively studied and are relatively well understood. 2 Dinuclear metallohydrolases have been studied to a lesser extent. 1 It is often proposed that a bridging hydroxide ion, which occurs in numerous dinuclear enzymes, serves as a nucleophile that attacks metal-bound substrates. 2,3 The low pK a value for the bridging water at dinuclear sites results in a higher local concentration of hydroxide ion compared to that afforded at mononuclear centers. However, simultaneous coordination of hydroxide to two metal ions also results in tighter binding and lowered nucle...

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

“…Lippard and coworkers reported a Zn(II) complex with a bridging hydroxide where the nucleophile was an external water ( pK a = 7.06) activated by the bridging hydroxide as base. 146 The pK a of 10.1, however, would be unusually high for a Cd(II)-bound alcohol. An 18 O-labeling experiment suggests that the nucleophile in OP hydrolysis by [Cd 2 (CO 2 EtH 2 L1)(CH 3 COO) 2 ] + is not a ligand centred alkoxide but rather a water molecule which is terminally bound to one Cd(II) ion.…”

Biomimetic Hydrolysis of Penicillin G Catalyzed by Dinuclear Zinc(II) Complexes: Structure–Activity Correlations in β‐Lactamase Model Systems

“…[30] While several groups have dealt with the hydrolysis or methanolysis of b-lactams by using simple transition-metal salts including Zn 2 + salts, [31,32] very few studies have employed preorganized dinuclear zinc(ii) complexes as functional mimics of metallo-b-lactamases. [30,33,34] Such models might provide valuable insights into the mechanistic role of the second Zn 2 + ion, cooperative effects of the adjacent metal ions, and the identity of the active nucleophile. Lippard and co-workers recently reported an in-depth study of nitrocefin hydrolysis mediated by phenol-and naphtyridinebased dinuclear zinc complexes.…”

“…Lippard and co-workers recently reported an in-depth study of nitrocefin hydrolysis mediated by phenol-and naphtyridinebased dinuclear zinc complexes. [33,34] From the observation that some mononuclear zinc(ii) complexes are as efficient in nitrocefin hydrolysis as the dinuclear zinc(ii) complexes they concluded that the second Zn 2 + is not required for catalytic activity. Depending on the specific system, either a bridging or a terminal hydroxide was found to act as the nucleophile.…”

“…Depending on the specific system, either a bridging or a terminal hydroxide was found to act as the nucleophile. [33,34] Preferential metal-ion coordination of b-lactam substrates by means of a monodentate carboxylate group was inferred from 13 C NMR and IR spectroscopy. [33,34] Here we report a comparative study on the hydrolytic cleavage of penicillin G (pen) mediated by the recently reported dinuclear zinc(ii) complexes 1-4, [35][36][37] as well as structural and spectroscopic insights into the mode of substrate binding.…”

“…[33,34] Preferential metal-ion coordination of b-lactam substrates by means of a monodentate carboxylate group was inferred from 13 C NMR and IR spectroscopy. [33,34] Here we report a comparative study on the hydrolytic cleavage of penicillin G (pen) mediated by the recently reported dinuclear zinc(ii) complexes 1-4, [35][36][37] as well as structural and spectroscopic insights into the mode of substrate binding. Complexes 1-4 are based on multifunctional pyrazolate ligands HL 1 -HL 4 , which offer the advantage to constrain the accessible range of metal-metal separation by means of the lengths of the chelating side arms attached to the heterocycle.…”