NMR studies of 111Cd6-MT 1 from lobster have been conducted to determine coordination structure of Cd-thiolate binding in the protein. Sequential proton resonance assignments were made using standard two-dimensional 1H NMR methods. Two-dimensional 1H-111Cd HMQC experiments were then carried out to determine the cadmium-cysteine connectivities in the protein. With this information, it was established that the six Cd ions exist in two different Cd3S9 clusters, each involving three bridging and six terminal thiolate ligands. Sequential cysteines in the sequence provide the sulfhydryl ligands for each cluster and do not overlap, as has been found in mammalian metallothionein. Comparison of the N-terminal, Cd3S9 B-type cluster of lobster MT 1 with the Cd3S9 cluster from rabbit MT 2 shows that while eight of the nine cysteine residues occupy homologous positions in their sequences, three of the 12 Cd-thiolate connectivities are different. Similarly, the C-terminal B-cluster of lobster MT 1 was compared with the Cd4S11 cluster of mammalian MT 2, excluding the two terminal cysteine sulfhydryl groups that convert this cluster from A- to B-type. As above, eight of nine cysteine positions are identical, yet five of 12 Cd-sulfhydryl connections are different. These differences are expanded when the role of each cysteine as bridging or terminal ligands in the clusters is considered.

The selenium-containing ester p-nitrophenyl (phenylselenyl)acetate, C6H5SeCH2C(O)-OC6H4-p-(NO2), has been synthesized, characterized as a substrate for alpha-chymotrypsin (k2/KM = 15.2 X 10(3) M-1 s-1, KMapp = 5.16 X 10(-6) M, pH 7.77, 33% CH3CN, 25 degrees C), and shown to be an active-site titrant for the enzyme. A synthesis of the selenium-77 enriched p-nitrophenyl (phenylselenyl)acetate in 53% yield from 94.4% elemental selenium-77, followed by its reaction with alpha-chymotrypsin (pH 5.0, 0-3 degrees C), permitted the observation of the (phenylselenyl)acetyl-alpha-chymotrypsin reaction intermediate by selenium-77 NMR spectroscopy. This acyl-enzyme species had a chemical shift of 275.1 ppm relative to dimethyl selenide. Accompanying this resonance was a lower intensity, pH-dependent resonance that is assigned to (phenylselenyl)acetate on the basis of a pH titration of the model compound. Deacylation in the presence of hydrazine sulfate produced a resonance at 332.3 ppm in addition to the 302.2 ppm resonance of (phenylselenyl)acetate at pH 7.85. Denaturation of the acyl-enzyme resulted in a shift of the 275.1 ppm resonance to 334.6 ppm at pH 4.90, in good agreement with the selenium-77 chemical shift of the model compound, methyl (phenylselenyl)acetate, in CDCl3 (333.3 ppm). The large shielding observed for the native acyl-enzyme in comparison to the denatured species can be attributed to a resonance-perturbed ester linkage and/or steric compression at a nonbonding orbital of the selenium nucleus.