| Abstract: | The inhibition of papain by N-acetyl-D- and N-acetyl-L-phenylalanyl[1-13C]glycinal was investigated by 13C nuclear magnetic resonance (NMR) spectroscopy. Both the L- and D-aldehyde enantiomers formed thiohemiacetals with papain. The 13C-enriched carbon of the thiohemiacetals formed with the L- and D-aldehydes has chemical shifts at 74.7 and 75.1 ppm, respectively. The difference in chemical shift for the two inhibitor complexes is attributed to each forming a different diastereomeric papain thiohemiacetal. Each enantiomeric inhibitor formed two diastereomeric thiohemiacetals with chiral thiols but produced a single diastereoisomer with papain. It is concluded that with papain thiohemiacetal formation is stereospecific. The D inhibitor is bound only 5-fold less tightly than the L inhibitor, which suggests that in both these inhibitor complexes the phenyl ring of the inhibitor phenylalanine is bound at the S2 hydrophobic pocket of papain. This is supported by computer modeling studies that show that both the N-acetyl-D- and N-acetyl-L-phenylalanine moieties can be separately fitted into the S2 subsite with the phenyl ring of phenylalanine in the S2 hydrophobic pocket. It is concluded that thiohemiacetal formation at S1 (S1 and S1' are the active center amino acid binding sites) is stereospecific with both D and L inhibitors. Computer modeling studies support this showing that, due to steric hindrance between the thiohemiacetal hydroxyl group and the backbone amide nitrogen of serine-24, only one of the two possible thiohemiacetal enantiomers can be formed at the S1 subsite. The thiohemiacetals formed from both the D- and L-aldehyde inhibitors therefore have only one permitted conformation at S1.(ABSTRACT TRUNCATED AT 250 WORDS) |
