Development of a method for the incorporation of substitution-inert metal ions into proteins. Site-specific modification of arsanilazotyrosine-248 carboxypeptidase A with cobalt(III)

Abstract

This investigation demonstrates the use of substitution-inert metal ions as site-specific amino acid modifying reagents. The approach involves the production of a chelating agent at the site of interest with the subsequent in situ oxidation of substitution-labile cobalt(II) to exchange-inert cobalt(III) with H2O2. We have produced the chelate complex ethylenediamine-N,N'-diacetato(arsanilazotyrosinato-248 carboxypeptidase A)cobalt(III) [CoIII(EDDA)(AA-CPA-Zn)]. Model CoIII(EDDA)(azophenolate) complexes have helped to define the reaction conditions necessary to produce the enzyme derivative and have proved invaluable in the spectral analysis of the cobalt(III)-enzyme complex. The modified enzyme contains one active-site zinc and one externally bound cobalt per enzyme monometer. Circular dichroism and visible spectra of the derivative and apoenzyme substantiate the site-specific nature of the incorporation. Concimitant with CoIIIEDDA incorporation, the enzyme loses its peptidase activity yet maintains with FeIIEDTA returns the original properties of the arsanilazotyrosine-248 enzyme.

Formation and Characterization of an Active Phosphoenolpyruvate Carboxykinase−Cobalt(III) Complex

“…Cobalt salts have been reported to promote the oxidation and destruction of the protein cytochrome P-450 and GSH (Moorhouse et al, 1985). Metalcatalyzed free radical damage was detected during the Co 3+ modification of arsanilazotyrosine-248 carboxypeptidase A (Urdea & Legg, 1979). The addition of free radical scavengers such as phenol prevented the free radical oxidative damage.…”

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

“…All kinetic experiments were performed at 25 OC. The wavelength used in following the various reactions were 220-236 nm for CBZ-Gly-Phe, 250-258 nm for HPLA, and 281-289 nm for HPA (Whitaker et al, 1966;Urdea & Legg, 1979;Davies et al, 1978). Infinity values were determined for each reaction solution in order to check the stoichiometry.…”

Indirect metal-metal linkage: cyclic ferrocene complexes with a second metal linked via remote phosphine functionality

“…Significantly, these cyclopentadienes are completely inert with respect to Diels-Alder dimerization, a quality shared with (trialkylsilyl)cyclopentadienes in general and a potential advantage over related systems possessing less bulky substitution on the ring. Deprotonation of crude mixture 7 in petroleum ether immediately results in the separation of 1 from solution as a gummy semisolid. Depending on the nature and quantity of the impurities present, it is often (but not always) possible to reduce this to a white solid by repeated vigorous washings with petroleum ether to remove possible contaminants such as 5 and 6.…”

“…The purified ferrocene is air stable both in the absence of solvent and in solution and exhibits spectral characteristics (2) c6d6 4.12 ("t", 1.8), 4.32 ("t", 1.7) 0.34 (d, 0.9) 1.66 (d, 0.6) 7.08-7.34 (m) CDC13 3.98 (m), 4.23 (m) 0.14 (br s) 1.50 (br s) 7.14-7.70 (br m) 2-dioxide (13) CDC13 3.93 ("t", 1.8), 4.16 ("t", 1.8) 0.28 (br s) 1.82 (d, 15) 7.2-7.9 (m) 2-Mo(CO)4 (15) c6d6 3.9-4.5 (m) 0.03, 0.18, 0.31 (s) 1.95-2.45 (br m) 7.0-8.1 (m) 2-Mn(CO)3Br (16) cdci36 3.82 (brs),c 4.14 (br s),c -0.13 (s) 2.32°2.52 (br s) 7.33 (br s), 7.64 (brs)…”

“…[[Diinethyl((diphenylphosphino)methyl)silyl]cydopentadienyl]lithium (1). A solution containing the above mixture of isomers (ca.…”