Carboxypeptidase metal substitution

Even though this dipeptide is turned over quite slowly, the complex examined is probably a non-productive one. Furthermore an analogous ester substrate has not been found, and it is known that carboxypeptidase behaves quite differently toward ester and peptide substrates. In particular, the kinetic parameters for peptide hydrolysis for a series of metal substituted carboxypeptidases indicate that fccat values can range from 6000 min for the cobalt enzyme down to 43 min for the cadmium enzyme 66). The values on the other hand are almost totally independent of the particular metal present. The exact opposite is true for ester hydrolysis. Km varies from 3300 M for the cobalt enzyme to 120 M for the cadmium enzyme while k<.at is essentially unchanged. 

Cobalt has recently been used as an ESR active substitute in zinc metalloenzymes. Whilst liquid helium temperatures may be needed and theoretical aspects of the spectra are not yet as well understood, cobalt has two important advantages over copper as a metal substitute, namely that many cobalt derivatives show some enzymic activity (e.g. cobalt in carbonic anhydrase, alkaline phosphatase and superoxide dismutase) and that g values and hyperfine splitting are more sensitive to ligand environment, particularly when low spin. ESR data have been reported for cobalt substituted thermolysin, carboxypeptidase A, procarboxypeptidase A and alkaline phosphatase [51]. These are all high spin complexes. Cobalt carbonic anhydrase has been prepared and reacted with cyanide [52]. In... 

The introduction of redox activity through a Co11 center in place of redox-inactive Zn11 can be revealing. Carboxypeptidase B (another Zn enzyme) and its Co-substituted derivative were oxidized by the active-site-selective m-chloroperbenzoic acid.1209 In the Co-substituted oxidized (Co111) enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Oxidation of the native enzyme resulted in modification of a methionine residue instead. These studies indicate that the two metal ions impose different structural and functional properties on the active site, leading to differing reactivities of specific amino acid residues. Replacement of zinc(II) in the methyltransferase enzyme MT2-A by cobalt(II) yields an enzyme with enhanced activity, where spectroscopy also indicates coordination by two thiolates and two histidines, supported by EXAFS analysis of the zinc coordination sphere.1210... 

Co11 complexes of imidazole retain some interest as structural models for biological systems, notably carboxypeptidase A and thermolysin. Vallee and colleagues discuss this with special reference to MCD spectra,322 and Hodgson discusses stereochemical aspects of purine and metal-nucleotide complexes for similar reasons.323 Horrocks, Ishley and Whittle have recently described some [Co(02CR)2(im)2] structural models, e.g. (61), which have physical properties similar to Co"-substituted carboxypeptidase.324... 

The substitution of another metal for that present in the native state or the removal of any metal is the simplest chemical modification for a metalloenzyme. Marked changes in activity are usually observed in either case. Substitution of cadmium for zinc first demonstrated a difference in the esterase and peptidase activities of carboxypeptidase A (47). The activity of [(CPD)Cd] toward Bz-Gly-L-OPhe is increased, but that enzyme is virtually inactive toward Cbz-Gly-L-Phe. 

Numerous metalloenzymes have the ability to remain functional even after the metal, which presumably is present at their active center, has been replaced by another metal (13). Thus in zinc deficiency, if the apoenzyme is synthesized, as has been observed in the case of . coli alkaline phosphatase (13), then other metals which might have accumulated or are normally within the cell could substitute for zinc and generate an active enzyme. Although this is a possibility in the case of microorganisms, it certainly does not appear to be true in the case of experimental animals and man, in that the apoenzymes of alkaline phosphatase, carbonic anhydrase, carboxypeptidase, alcohol dehydrogenase, and de-oxythymidine kinase do not accumulate in zinc-deficient tissues. Thus, one may conclude that a deficiency of zinc does specifically aflFect the activities of zinc-dependent enzymes in sensitive tissues. 

Circular dichroism (CD) has played an important role in our studies on the modification of enzymes and hormones with Co(III). The objective of these studies has been to incorporate selectively substitution inert metal ions at specifically modified sites in proteins as probes of biological function. Significant information concerning the catalytic mechanism of carboxypeptidase A (CPA) (1) has been obtained from a site specific modification of tyrosine 248 with Co(III) (2). The method developed for CPA has been extended to other enzymes and hormones in order to devg op an improved method for incorporating stable radioisotopes t Co) into proteins. The substitution-inertness of Co(III) provides the necessary stability in these derivatives (3). 

Enzymes having metals as their components can also be inhibited by a substitution of one of these metal ions by another ion with the same charge and a similar size. For example, the toxic effect of cadmium is due to a substitution for zinc, which is a common component in metalloenzymes. The Zn " " and Cd ions are chemically similar, however, the cadmium-containing enzyme does not function properly. The Cd " ions can result, for instance, in the inhibition of amylase, adenosine triphosphatase, adcohol dehydrogenase, glutamic-oxalacetic transaminase, carbonic anhydrase and peptidase activity in carboxypeptidase [4]. 

The investigation of divalent metal ion-substituted carboxypeptidase A derivatives has shown that the apparent pKa of the high pH ionization is independent of the metal ion. In contrast, the apparent pKa of the low pH ionization changes from 6.33 for the zinc(II) enzyme to 5.57 for the cobalt(II) enzyme when a-N-benzoyl-GlyGly-L-Phe is used as the substrate 151). Although this pH dependence strongly implies that the metal ion directly influences the low pH ionization, the visible spectrum of the cobalt(II) enzyme is not perturbed by this ionization 166). The spectrum of the cobalt(II) enzyme, however, is pH-dependent above pH 8.0. The spectral changes titrate with an apparent pKa of 8.8 166). Note that this value is approximately the same as the apparent pKa which is reflected in the pH-dependence of Am (Fig. 10 c). 

Although other probes may be substantially easier to use, XAS is sometimes the only method that is sensitive to the structures of interest, particularly for solid-state samples and in situ studies of catalysts.For example, in a study of the reduction of NiO, time-resolved X-ray diffraction had shown that the catalyst went directly from NiO Ni without a well-ordered intermediate phase, but could not rule out the existence of an amorphous NiO phase, since the diffraction was not sensitive to disordered phases. However, the formation of an intermediate phase could be ruled out by time-resolved EXAFS. A similar situation exists for spectroscopically silent metals systems), which are difficult to probe with methods other than XAS. Examples that are important in bioinorganic chemistry include Cu and Zn. For carboxypeptidase, time-resolved XAS could be used to determine the rate of change of the native site, while conventional UV-visible methods could only be used on the Co " " substituted enzyme. The importance of XAS in both of these examples is as a tool for measuring rate constants. 

Transition metal complexes of optically active ligands display ORD and CD curves in the region of the metal d— d absorption region. Substitution of Co " for in carboxypeptidase provides a "spectroscopic probe". CD studies of the Co " d— d transitions provide useful information about the environment at the metal ion site. 

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