Metal ion effects in the hydrolysis of esters and anhydrides

Abstract Carboxypeptidase A is a Zn(II) metalloenzyme which catalyzes the hydrolysis of ester and peptide substrate [1]. X-ray crystallographic analysis at 2-A resolution has shown the zinc ion to be chelated to the carbonyl oxygen of poor peptide substrates [1–3]. The carboxyl group of glutamic acid-270 has also been implicated in the catalytic process, and mechanisms have been suggested involving nucleophilic attack and classical general base catalysis [2, 3]. Evidence has been presented for a nucleophilic mechanism in the enzyme catalyzed hydrolysis of esters at low temperatures [4], the reaction presumably proceeding via an anhydride intermediate. It has therefore been a great importance to determine the effects of metal ions in ester hydrolysis reactions involving carboxyl group participation and in the hydrolysis of anhydrides. Divalent metal ions (Cu 2+ , Zn 2+ , Ni 2+ , and Co 2+ ) at saturating concentrations produce large rate enhancements in the hydrolysis of esters (ranging up to 10 8 ) via a metal ion promoted attack of hydroxide ion [5–7]. When the living group is phenolic, mechanisms involving intramolecular carboxyl group participation, either as a nucleophile or a general base, cannot compete effectively with the metal ion promoted hydroxide ion reaction [5, 6]. This is because carboxyl group participation in these systems is not appreciably enhanced by the metal ions. However, when the living group is an aliphatic alcohol of high pK a , then significant rate enhancement are observed in carboxyl nucleophilic reactions [7, 8]. In the hydrolysis of 2-(6-carboxypyridyl)methyl hydrogen phthalate, saturating concentrations of Co 2+ and Ni 2+ enhance the rate of intramolecular carboxyl attack over 100 fold while with Cu 2+ the rate enhancement is 10 4 [7]. In these reactions the metal ion exerts its effect through a transition state effect in which the leaving group is stabilized. This appears to be a general mechanism in reactions in which CO bond breaking is rate determining. (See next column) Both metal ion promoted water and OH − catalyzed reactions are observed in the hydrolysis of cinnamic (6-carboxy)picolinic monoanhydride [9]. Rate enhancements in the former reaction are greater than 100 fold at saturating concentrations of Ni 2+ , Co 2+ , and Zn 2+ . The rate constants in these reactions are only approximately 100 fold less than in comparable reactions in the Zn(II), Ni(II), and Co(II) carboxypeptidase A catalyzed hydrolysis of the ester substrate O-( trans -cinnamoyl)-L-β-phenyllactic acid. The Zn(II), Ni(II), and Co(II) carboxypeptidases give similar plots of k cat , K m , and k cat /K m vs. pH in hydrolysis of O-( trans -cinnamoyl)-L-β-phenyllactic acid in H 2 O at 30 °C. The k cat vs. pH profiles all show a sigmoidal region in which pK ES app values are closely similar (Zn(II) 6.2; Ni(II) 6.2., and Co(II) 5.7). At pH > 9 apparent OH − catalyzed reactions occur with rate enhancements of 10 7 – 10 8 over nonenzymatic OH − catalyzed hydrolysis of the ester. These reactions very likely represent metal ion promoted OH − catalyzed breakdown of the anhydride intermediate similar to the reactions observed in the model studies. Modification of the carboxyl group of Glu-270 to the methoxyamide by the method of Petra [10] leads to loss of activity at all pH values including pH > 9. It is probable that breakdown of an anhydride intermediate is rate determining at all pH values greater than 6. Both formation and breakdown of the anhydride intermediate are very likely facilitated by the metal ion.