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Titolo:
MECHANISTIC STUDIES ON THE AMINOPEPTIDASE FROM AEROMONAS-PROTEOLYTICA- A 2-METAL ION MECHANISM FOR PEPTIDE HYDROLYSIS
Autore:
CHEN GJ; EDWARDS T; DSOUZA VM; HOLZ RC;
Indirizzi:
UTAH STATE UNIV,DEPT CHEM & BIOCHEM LOGAN UT 84322 UTAH STATE UNIV,DEPT CHEM & BIOCHEM LOGAN UT 84322
Titolo Testata:
Biochemistry
fascicolo: 14, volume: 36, anno: 1997,
pagine: 4278 - 4286
SICI:
0006-2960(1997)36:14<4278:MSOTAF>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
LENS LEUCINE AMINOPEPTIDASE; TRANSITION-STATE-ANALOG; PURPLE ACID-PHOSPHATASE; X-RAY CRYSTALLOGRAPHY; CRYSTAL-STRUCTURE; ACTIVE-SITE; ZINC ENZYMES; METHIONINE AMINOPEPTIDASE; KLEBSIELLA-AEROGENES; CARBOXYPEPTIDASE-A;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
55
Recensione:
Indirizzi per estratti:
Citazione:
G.J. Chen et al., "MECHANISTIC STUDIES ON THE AMINOPEPTIDASE FROM AEROMONAS-PROTEOLYTICA- A 2-METAL ION MECHANISM FOR PEPTIDE HYDROLYSIS", Biochemistry, 36(14), 1997, pp. 4278-4286

Abstract

The aminopeptidase from Aeromonas proteolytica (AAP) is uncompetitively inhibited by fluoride ion at pH 8.0 with an inhibition constant (K-i) of 30 mM. Thus, fluoride inactivates AAP only after substrate binding, and only a single fluoride ion binds to AAP. On the other hand, chloride ion does not inhibit AAP up to concentrations of 2 M at pH 8.0. The pH dependence of fluoride inhibition of AAP was measured over thepH range 6.0-9.5. Between pH values of 6.0 and 9.0, fluoride ion actsas a pure uncompetitive inhibitor of AAP, and the K-i increases from 1.2 to 370 mM. From a plot of pK(i) vs pH, a pK(a) value of 7.0 +/- 0.3 was extracted which corresponds to a single deprotonation process. At pH values higher than 9.0, the fluoride inhibition pattern changes to competitive. This change in inhibition pattern was attributed to a change in ionic strength or perhaps pH of the solution since fluoride ion was also found to become a competitive inhibitor of AAP at pH 8.0 in the presence of 2 M NaCl. These data, taken together with previous kinetic studies of mono- and dinuclear hydrolases with fluoride ion, suggest that a Zn(II)-bound water/hydroxide exists at the dimetal activesite of AAP with a pK(a) of 7.0 and that this water/hydroxide acts asthe active site nucleophile. The hydrolysis of L-leucine-p-nitroanilide was measured spectrophotometrically in triplicate between 25 and 85degrees C at eight substrate concentrations ranging from 5 to 800 mu M. From these data, K-m values were derived at each temperature studied and were found to increase exponentially with increasing temperature. Moreover, the calculated V-max values were also found to increase over this temperature range, mimicking the Km values. An Arrhenius plot was constructed from k(cat) values and was found to be linear over thetemperature range 25-85 degrees C, indicating that the rate-limiting step in AAP peptide hydrolysis is product formation and does not change as a function of temperature. From the slope of the line, the activation energy (E(a)) was calculated to be 36.5 kJ/mol, The enthalpy and entropy of activation at 25 degrees C calculated over the temperature range 298-358 K were found to be 34.0 kJ/mol and -94.2 J/(mol . K), respectively. The free energy of activation at 25 degrees C was found tobe 62.1 kJ/mol. Combination of the available X-ray crystallographic data with the present kinetic and thermodynamic results, as well as thepreviously reported kinetic and spectroscopic data, has allowed a detailed catalytic mechanism for AAP to be proposed.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 01/12/20 alle ore 19:41:39