Enzyme-bound visible spectra

Little is known about the mechanism of action of the purple acid phosphatase. The beef spleen enzyme as isolated contains one tightly bound phosphate,821 but it is not certain whether this corresponds to a phosphorylated amino acid residue as found for other phosphatases. Addition of phosphate causes a shift in the visible spectrum of the enzyme. 

An early remarkable finding in one of the crystal structure studies was that the product, dimethylsulfide (DMS), could be added to the oxidized enzyme to reduce the active site in the reverse of the physiological reaction. This product-reduced enzyme showed remarkable changes in the UV visible spectrum, and DMSO was observed crystallographically to be present in the active site, bound to Mo, although the structural conclusions may have suffered from the same multiplicity of active site structures that we have discussed above. The DMS-reduced enzyme was unambiguously shown by XAS to be a... 

The ribonucleotide reductase of Lactobacillus leichmanii catalyses the rapid conversion of enzyme-bound coenzyme B12 to an intermediate which has an absorption spectrum in the visible and u.v. like that of cob(ii)alamin (Biar)- Coffman et al. have recently analysed the e.s.r. spectrum of this intermediate and suggest that it contains low-spin cobalt(ii) with strongly distorted six-fold co-ordination. 

The fully reduced state of CCP is probably of no physiological significance. However, the visible region spectrum of the fully reduced enzyme is very sensitive to the presence or absence of bound Ca + ions. In the absence of Ca + ions the a-band maximum of the reduced enzyme, recorded during the course of the redox titrations described earlier, is at 551 nm with a shoulder at 557 nm. In contrast, in fully reduced Ca +-loaded enzyme the maximum is at 557 nm with a shoulder 551 nm (52). These spectra should be compared with those of the fully reduced Psew-domonas CCP in its inactive and active forms, respectively, which show similar differences (81). Further similarities exist between the X-band EPR spectra of the active CCP from P. aeruginosa in the MV state and that of the Ca +-loaded CCP from P. denitrificans and, moreover, the X-band EPR spectrum of MV P denitrificans CCP that had previously been treated with EDTA (Table I). 

The purple acid phosphatases can occur in two diferric forms—one as the tightly bound phosphate complex (characterized for the bovine and porcine enzymes) (45, 171, 203) and the other derived from peroxide or ferricyanide oxidation of the reduced enzyme (thus far accessible for only the porcine enzyme) (206). These oxidized forms are catalytically inactive. They are EPR silent because of antiferromagnetic coupling of the two Fe(IIl) ions and exhibit visible absorption maxima near 550-570 nm associated with the tyrosinate-to-Fe(III) charge-transfer transition. The unchanging value of the molar extinction coefficient between the oxidized and reduced enzymes indicates that the redox-active iron does not contribute to the visible chromophore and that tyrosine is coordinated only to the iron that remains ferric in agreement with the NMR spectrum of Uf, (45). 

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