Flexible loops, often referred to as flaps, have been shown to play a role in catalytic mechanisms of different enzymes. Flaps at the active site regions have been observed in the crystal structures of aspartic proteinases and their residues implicated in the catalytic processes. This research investigated the role of the flap residue, threonine 77, in the activation of pepsinogen and the catalytic mechanism of pepsin. Three mutants, T77S, T77V and T77G, were constructed. Differences in amino acid polarity and hydrogen bonding potential were shown to have an influence on the activation and catalytic processes. T77S activated at the same rate and had similar catalytic parameters as the wild-type pepsin. The activation rates of T77V and T77G were slower and their catalytic efficiencies lower than the wild-type. The results demonstrated that the threonine 77 polar side chain played a role in a proteolysis. The contribution of the side chain to zymogen activation was associated with the proteolytic cleavage of the prosegment. It was postulated that the hydroxyl group at position 77 provided an essential hydrogen bond that contributed to proper substrate alignment and, indirectly, to a catalytically favorable geometry of the transition state.

Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71--80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes.

No abstract

Glycine residues are known to contribute to conformational flexibility of polypeptide chains, and have been found to contribute to flexibility of some loops associated with enzymic catalysis. A comparison of porcine pepsin in zymogen, mature and inhibited forms revealed that a loop (a flap), consisting of residues 71-80, located near the active site changed its position upon substrate binding. The loop residue, glycine-76, has been implicated in the catalytic process and thought to participate in a hydrogen-bond network aligning the substrate. This study investigated the role of glycine-76 using site-directed mutagenesis. Three mutants, G76A, G76V and G76S, were constructed to increase conformational restriction of a polypeptide chain. In addition, the serine mutant introduced a hydrogen-bonding potential at position 76 similar to that observed in human renin. All the mutants, regardless of amino acid size and polarity, had lower catalytic efficiency and activated more slowly than the wild-type enzyme. The slower activation process was associated directly with altered proteolytic activity. Consequently, it was proposed that a proteolytic cleavage represents a limiting step of the activation process. Lower catalytic efficiency of the mutants was explained as a decrease in the flap flexibility and, therefore, a different pattern of hydrogen bonds responsible for substrate alignment and flap conformation. The results demonstrated that flap flexibility is essential for efficient catalytic and activation processes.

A crystalline food protein isolated from dried seeds of white kidney beans (Phaseolus vulgaris) was investigated for the presence and characteristics of phaseolin polypeptides. Reversed-phase highperformance liquid chromatography (RP-HPLC) of the crystalline protein gave five fractions, of which the first two eluting fractions (F1 and F2) were found to contain phaseolin polypeptides. Ion-spray mass spectrometry showed that the average molecular weights (MW) for F1 and F2 were 49 615 and 48 075. Amino acid composition and N-terminal sequence analysis indicated that the fractions F1 and F2, separated from the crystalline protein, contained polypeptides which were similar to those reported for a-type and &type phaseolin precursors, respectively.