Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Abstract

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Mechanistic studies of the astacin-like Serratia metalloendopeptidase serralysin: highly active (>2000%) Co(II) and Cu(II) derivatives for further corroboration of a "metallotriad" mechanism

“…A few Cu 2+ -substituted Zn hydrolases, including Aeromonas aminopeptidase (aAP) [48], astacin [53], and serralysin, also exhibit significant activities, which further corroborates the theory. Di-Cu-aAP exhibits only 5% and 9%, respectively, activities in terms of k cat and k cat /K m toward the hydrolysis of the specific substrate Leu-pNA [48], while Cu 2+ -astacin shows 37% activity in terms of k cat /K m toward succinyl-tri-Ala-pNA hydrolysis [53]. Despite the high Lewis acidity of Cu 2+ , almost all Cu 2+ -substituted derivatives of Zn hydrolytic enzymes are inactive or exhibit much lower activities than the native enzymes [32,44].…”

“…Consequently, several synthetic Cu 2+ complexes with high Lewis acidity have been demonstrated to catalyze hydrolytic reactions [45,46,47]. A few Cu 2+ -substituted Zn hydrolases, including Aeromonas aminopeptidase (aAP) [48], astacin [53], and serralysin, also exhibit significant activities, which further corroborates the theory. Di-Cu-aAP exhibits only 5% and 9%, respectively, activities in terms of k cat and k cat /K m toward the hydrolysis of the specific substrate Leu-pNA [48], while Cu 2+ -astacin shows 37% activity in terms of k cat /K m toward succinyl-tri-Ala-pNA hydrolysis [53].…”

“…This has been attributed to the similar coordination chemistry of Co 2+ and Zn 2+ [44,49]. However, the much higher activity of Co 2+ -serralysin than that of the native enzyme (Table 1) is still seldom seen among all Co 2+ -substituted Zn enzymes [44,48,49,50,51,53]. It is worth noting that Co-serralysin also exhibits a nearly full endopeptidase activity (81%) toward denatured casein (Fig.…”

“…Therefore, the highly active Cu-serralysin can serve as a prototype to gain further insight into serralysin catalysis and to provide more information about Cu 2+ -centered hydrolytic reactions. Conversely, full or higher activities are usually observed for Co 2+ -substituted derivatives of Zn 2+ enzymes [44,49], such as aminopeptidase [48,50], carboxypeptidase A [51], and astacin [53]. This has been attributed to the similar coordination chemistry of Co 2+ and Zn 2+ [44,49].…”

Structural Basis for Substrate Recognition and Hydrolysis by Mouse Carnosinase CN2

“…Mutations in Tyr 246 of S. griseus aminopeptidase and Tyr 552 of glutamate carboxypeptidase G II, which are located in the position of His 228Ј in CN2, resulted in about 100-and 10-fold reduction of activity, respectively (41,42), indicating that the residues are essential in the hydrolysis reaction in these peptidases. In the crystal structure of PepV in complex with a phosphinic inhibitor Asp⌿[PO 2 (7,44). A comparison of the active center of CN2 with those of M28 metallopeptidase family proteins revealed that the ligand residues and catalytic Glu are located in the same positions.…”