| Hyperthermophiles were the first life forms to have arisen on earth. Hyperthermophilic enzymes from these archaea and bacteria can therefore serve as ideal model systems in understanding enzyme evolution molecular mechanisms for protein thermostability, and the relationship between structure and function.The enzyme APE1547 from the thermophilic archaeon Aeropyrum pernix K1 was characterized as acylpeptide hydrolase and possessed both acylpeptide hydrolase and esterase activity. Previous study indicated that APE1547 showed extremely high thermalstability and denaturant resistance. The crystal structure indicates that APE1547 containes two functional domains, which are the N-terminal contains aβ-propeller with seven blades and the C-terminal contains a canonicalα/βhydrolase fold with a central eight-strand mixedβ-sheet. A shortαhelix at the N-terminal extends from theβ-propeller domain and forms part of the hydrolase domain. With the sequence alignment, we found that APE1547 belongs to prolyl oligopeptidase family. The possible role of the N-terminal extension is believed to take the role of control the entrances of substrate to the catalytic center.The enzyme was presumed to be most like the acylamino acid-releasing enzyme (AARE) by the crystal structure and sequence alignment. Therefore, its character of AARE, the effect of amino acids and metal cations were examined. The mechanism of how calcium cation affects the activity of the enzyme was study by fluorescence and circular dichroism simply. From these results, the enzyme was considered to be a bifunctional enzyme like other enzymes reported. It had the activity of esterase and acylamino acid-releasing enzyme.The enzyme was obtained from engineering bacterium constructed by Dr. Gao and purified and determined according to the procedures described in the Gao's dissertation. The optimal AARE activity of APE1547 was detected at 90℃, like its esterase activity. The optimal pH for the substrate Ac-Leu-pNA was 8.0 which same to its esterase and for the peptide substrates was 7.25. The pH discrepancy may be caused by the distinctness in enzyme assay method of these substrates. Among the small peptides, f-Met-Ala was the optimum substrate and kinetic parameters of AARE were 0.88±0.02 mM, 1.959±0.425 S-1 for Km and kcat respectively. The effect of metal cations on the enzyme was very complex: (1) calcium, magnesium and barium ions had stimulative effect of AARE; (2) zinc, manganese, sodium ions inhibited the activity of AARE and (3) copper ion resulted in complete inactivation of enzyme. Amino acids had no significant inhibition to the activity of AARE and had no effect of esterase activity. The performance of these amino acids was much like product inhibition rather than inhibitor inhibition. The results may demonstrate that the enzyme was an AARE, although it had esterase activity. Like the thermostability of esterase, The AARE also had high thermostability and protein concentration-dependent. When the enzyme (0.8mg/ml) was incubated at 90℃for 36h, it still could retain approximately 50% of its original activity. Even though the enzyme concentration was low to 0.2mg/ml, the half-life was 16 h. It was easy to understand because the enzyme from the hyperthermophilic archaeon that grows optimally in high temperature.Calcium cation had bi-modal effect of AARE, it could activate enzyme activity when the ion concentration was lower than 0.2 M, and when the concentration of calcium ion was 0.1 M, and the enzyme had the highest activity. But when the concentration was higher than 0.2 M, the enzyme activity was inhibited. The intrinsic fluorescence of the enzyme was founded could be quenched by addition of the calcium cation. Recurring to the equation Stern-V?lmer, the calcium cation was bound to the enzyme and the mechanism of fluorescence quenching belongs to static quenching. From the calculation of fluorescence quenching results, association constant was 1.24x103 M-1, one enzyme molecule may be bound by one calcium cation. The relationship between concentration-dependent variety in the enzyme activity and the change in the secondary structure was studied by circular dichroism spectroscopy. The activity enhanced by addition of calcium cation could result from the increase ofβ-turn and decrease of random coil. However, change ofα-helix content doesn't seem to associate with the enzyme activity. But where was the calcium cation in the enzyme molecule and how to interact with it leading to the change of enzyme activity should need to do further study.To study the function ofβ-propeller domain and catalytic domain, we cloned and expressed these two domains separately. We also characterized and investigated the stability of each domain.The experiment showed that theβ-propeller domain could be expressed in E. coli in soluble form and showed no activity after purified. The thermostability ofβ-propeller domain was determined tryptophan fluorescence; the results indicated that theβ-propeller domain still showed high thermostability. Theβ-propeller domain is very important to the hyperthermostability of APE 1547. For no activity center, it did not show catalytic function.Withoutβ-propeller domain, the recombinant catalytic domain was expressed in inclusion body form in E. coli, only few parts of them expressed in solution form. The whole molecular can be expressed in E. coli in soluble form; this means theβ-propeller domain might help the catalytic domain to fold in correct conformation. The single catalytic domain still showed enzyme activity, but the activity was really low compared to the whole molecular. This means the enzyme molecular needs theβ-propeller domain to keep its high activity. Structure study proved that some residues located in the surface ofβ-propeller domain and catalytic domain belongs to the activity center. The enzyme characterization proved that the optimum temperature was changed from 90 to 52 and optimum pH was shifted from 8.0 to 8.5; The suitable substrate is also pNPC8, but the substrate specificity was decreased, the catalytic domain was more suitable to long chain substrate. Thermostable research finds the stability of catalytic domain decreased sharply, the half time of catalytic domain at 50 was only 2 h, it totally inactivated within 1h at 60. All these results suggested thatβ-propeller domain is important to the whole molecular. This study indicated that the catalytic domain can take form the catalysis function separately, but the activity and stability was really low. Theβ-propeller domain takes on important role in helping molecular fold, keeping high activity and remaining good thermostability.
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