Aminopeptidases (APs) are a family of widely distributed proteases which participate in many significant biological processes, such as protein maturation, hormone production, and peptide digestion [1].   While several Zn peptidases are known to contain a single Zn2+ ion in their active site [2], a few metallo-APs, including those from bovine lens (bAP) [3],  Escherichia coli (eAP) [4],  Aeromonas proteolytica (aAP) [5],  and Streptomyces griseus (sAP) [6] have been proved by means of X-ray crystallography to contain a dinuclear metal active site.  However, diverse structural features and mechanistic properties have been revealed in these dinuclear metallo-APs [7];  including their different tertiary and quaternary structures, the lack of a conserved geometry and coordinated ligand type in the active site, and the observation of dinuclear catalysis in some APs while mononuclear catalysis in others.

    Despite the presence of a dinuclear site in aAP[5] and presumably in porcine kidney AP (pAP) [8],  these two APs have been observed to exhibit a "mononuclear" catalysis.  These two enzymes have been previously shown to exhibit a selective metal binding property, where a full hydrolytic activity was detected when one metal ion was bound [8,9]   A modulation of the activity was observed when a second metal ion was introduced, indicating a regulatory role [10].  Nevertheless, this mononuclear catalysis was not observed in bAP action [3].  Further studies of other dinuclear APs are therefore necessary to provide more mechanistic information about this mononuclear and dinuclear discrepancy in AP action.

    The AP isolated from the culture medium of Streptomyces griseus (sAP, MW ~30 kDa) has been characterized to contain 2 Zn2+ ions per molecule (see structure, and the active site structure shown below) [11].   A previous metal-activation study using the slow substrate Ala-p-nitroanilide revealed that Mn2+, Co2+, or Zn2+ was bound simultaneously to the two metal binding sites of the enzyme at pH 8 in the presence of 1 mM Ca2+; i.e., the activity of the enzyme was parallel to the amount of metal ion bound to the enzyme and reached a plateau with 2 equivalent metal ions introduced [11a].  Since the two metal sites could not be selectively filled with metal ions in the previous study, identification of each metal site was not possible and the role of each metal ion in catalysis could not be easily revealed.  It is important to find out the conditions for a selective metal binding to the metal sites, which is an inevitable step to provide structural information and catalytic role about each individual metal site by means of physical methods.

    We have been studying the metal binding properties, hydrolytic activity, and active site structure of sAP by means of activity assay and spectroscopic methods.  A sequential Co2+ binding to this enzyme in MES buffer at pH 6.1 has been concluded on the basis of optical study, isotropically shifted 1H NMR features, and activity assay.  Moreover, we have shown a very rare case that Cu2+ ion, which is better known as an NMR "relaxation probe", can be utilized as a "shift probe" for the study of metal binding sites in metalloproteins and affords sharp hyperfine-shifted 1H NMR signals (see abstract).  The presence of a dinuclear metal active site in sAP has been established by means of 1H NMR using Cu2+ as a probe.  This study also demonstrates that although sAP and aAP have nearly identical active sites on the basis of their crystal structures [5,6] (however, with a low sequence homology ~30% [12]), they exhibit quite different mechanisms in that sAP shows a dinuclear hydrolytic catalysis whereas aAP shows a mononuclear catalysis.

Our publications about Streptomyces aminopeptidase:

  1. Lin, L.-Y.; Park, H. I.; Ming, L.-J.* "Metal Binding and Active Site Structure of Di-Zinc Streptomyces griseus Aminopeptidase" J. Biol. Inorg. Chem. 1997, 2, 744-749.  (abstract and reprint in PDF)
  2. Holz, R. C.*; Bennett, B.; Chen, G.; Ming, L.-J. "Proton NMR Spectroscopy as a Probe of Dinuclear Copper(II) Active Sites in Metalloproteins.  Charaterization of the Hyperactive Copper(II)-Substituted Aminopeptidase from Aeromonas proteolytica" J. Am. Chem. Soc. 1998, 120, 6329-6335.  (abstract and reprint in PDF )
  3. Ming, L.-J. "NMR Studies of Paramagnetic Multinuclear Metalloproteins" Trends in Inorganic Chemistry 1998, 5, 205–236. (Table of contents and Introduction)
  4. Harris, M. N.; Ming, L.-J.* "Different Phosphate Binding Modes of Streptomyces griseus Aminopeptidase between Crystal and Solution States and the Status of Zinc-Bound Water"  FEBS Lett. 1999, 455, 321-324. (abstract and reprint in PDF)
  5. Park, H. I.; Ming, L.-J.* "A 1010 Rate Enhancement of Phosphodiester Hydrolysis by a Di-Zinc Aminopeptidase– Transition State Analogues as Substrates?" Angew. Chem. Intl. Engl. Ed. 1999, 38, 2914-2916. (abstract and reprint in PDF )
  6. Ercan, A.; Park, H. I.; Ming, L.-J.* "Remarkable Enhancement of the Hydrolyses of Phosphoesters by Dimetal Centers —Streptomyces Aminopeptidase as a “Natural Model System”" Chem. Commun. 2000, 2501–2502.  (abstract and reprint)
  7. Hasselgren, C.; Park, H. I.; Ming, L.-J.* "Metal Ion Binding and Activation of Streptomyces griseus Aminopeptidase —Cadmium(II) Binding as a Model" J. Biol. Inorg. Chem. 2001, 6, 120–127. (abstract and reprint)
  8. Harris, M. N.; Bertolocci, C.; Ming, L.-J.* "31P NMR Relaxation and Kinetic Studies of Cobalt(II)-Substituted Streptomyces Dinuclear Aminopeptidase" Inorg. Chem. 200241, 5582-5588. (abstract and reprint)
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