Oxidation of D-mannosamine (1), D-glucosamine (2), and D-galctosamine (3) by sodium N-chlorobenzenesulfonamide or chloramine-B (CAB) at 313 K is followed by a shortening of carbon chain and obeys the rate law, rate = k[CAB][sugar][HO(-)](x)(), where x is less than unity. The products are arabinonic acid, ribonic acid, and erythronic acid for 1 and 2 with smaller amounts of glyceric and hexonic acids, while lyxonic and threonic acids are predominant in the oxidation of 3 with smaller amounts of glyceric and hexonic acids. Proton inventory studies made in a H(2)O-D(2)O mixture point toward a single transition state. In the proposed mechanism the alkoxy anion (S(-)) of the hexosamine formed in a base-catalyzed reaction at C-1 carbon is subjected to an electrophilic rate-limiting attack by Cl(+) of the oxidant. The hexonic acid formed is decarboxylated with loss of ammonia to form the respective pentose, which is further converted into the corresponding pentonic acid. The breaking of the bond between C-1 and C-2 carbons in pentose yields tetronic acids. The thermodynamic parameters for sugar alkoxy anion formation and activation parameters for the rate-limiting step have been evaluated.

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Rice bran is a byproduct obtained from the rice milling industry, and to arrest lipolysis caused by lipolytic enzyme, rice bran lipase (RBL) was inactivated by inhibitors such as polyphenols. This study describes the inhibition and interaction of enzyme with chlorogenic acid (CGA) and caffeic acid (CA). The inhibition of the enzyme was competitive in nature in both CGA and CA. The inhibition constant K(i) of the reaction was found to be 1.8 and 1.5 muM for CGA and CA, respectively. Fluorescence emission measurements indicated a decrease in the fluorescence emission intensity and a red shift in the emission maximum as these ligands concentrations are increased, indicating the minor changes in the tryptophan environment and the effect of binding that is stronger in the case of CA compared to CGA with RBL. Far UV-circular dichroic data suggest that there are no significant changes in the conformation of the enzyme as a result of binding of CGA or CA. The instability of the enzyme in the presence of these polyphenols has been indicated by decrease in apparent thermal transition temperatures of the enzyme from a control value of 60 degrees C as revealed by thermal denaturation measurements. These results demonstrate that both CGA and CA are inhibitors of RBL and bind to the enzyme through both hydrogen and hydrophobic interaction in bringing about inhibition with minor structural alterations. These inactivation phenomena of polyphenols that act as inhibitors on RBL can be utilized to prevent oxidation of the rice bran oil.

Introduction:

The kinetics of inhibition of rice bran lipase (RBL) by phenylboronic acid (PBA) was studied to elucidate the nature of inhibition and the effect of the inhibitor on the structure-function of RBL. The effectiveness of an inhibitor is normally expressed by the constant K(i), which is calculated from the Lineweaver-Burk plot and found to be 1.7 mM at pH 7.4. The kinetics of inhibition by PBA was competitive, indicating the presence of serine in the active site of the enzyme. The loss of activity of RBL was concentration dependent on the inhibitor (PBA), and the inactivation followed a pseudo-first-order kinetics. Fluorescence emission measurements indicated a decrease in the fluorescence emission intensity and a red shift in the emission maximum as the inhibitor concentration was increased. The inhibition of the enzyme by PBA was also confirmed by thermal denaturation measurements, which indicated a shift in the thermal denaturation temperature of the enzyme toward lower temperatures. The far-UV-CD data suggest that there were no significant changes in the conformation of the enzyme as a result of binding of PBA. These results indicate that PBA is a potential inhibitor of RBL and binds to the enzyme in bringing about inhibition without any structural alterations.

The viral serpin, crmA, is distinguished by its small size and ability to inhibit both serine and cysteine proteases utilizing a reactive loop shorter than most other serpins. Here, we characterize the mechanism of crmA inhibition of serine proteases and probe the reactive loop length requirements for inhibition with two crmA reactive loop variants. P1 Arg crmA inhibited the trypsin-like proteases, thrombin, and factor Xa, with moderate efficiencies (∼10 2 -10 4 M −1 sec −1 ), near equimolar inhibition stoichiometries, and formation of SDS-stable complexes which were resistant to dissociation (k diss ∼10 −7 sec −1 ), consistent with a serpin-type inhibition mechanism. Trypsin was not inhibited, but efficiently cleaved the variant crmA as a substrate). N-terminal sequencing confirmed that the P1 Arg-P1ЈCys bond was the site of cleavage. Altering the placement of the Arg in a double mutant P1 Gly-P1ЈArg crmA resulted in minimal ability to inhibit any of the trypsin family proteases. This variant was cleaved by the proteases ∼10-fold less efficiently than P1 Arg crmA. Surprisingly, pancreatic elastase was rapidly inhibited by wild-type and P1 Arg crmAs (10 5 -10 6 M −1 sec −1 ), although with elevated inhibition stoichiometries and higher rates of complex dissociation. N-terminal sequencing showed that elastase attacked the P1ЈCys-P2ЈAla bond, indicating that crmA can inhibit proteases using a reactive loop length similar to that used by other serpins, but with variations in this inhibition arising from different effective P2 residues. These results indicate that crmA inhibits serine proteases by the established serpin conformational trapping mechanism, but is unusual in inhibiting through either of two adjacent reactive sites.

CrmA is a "cross-class" serpin family inhibitor of the proapoptotic serine protease, granzyme B, as well as cysteine proteases of the caspase family. To determine whether crmA inhibits these structurally diverse proteases by a common conformational trapping mechanism, we mapped the position of the protease in crmA complexes with granzyme B or caspase-1 by fluorescence perturbation and fluorescence resonance energy transfer (FRET) analyses of site-specific fluorophore-labeled crmAs. A reactive loop P6 NBD label underwent similar large fluorescence enhancements (>200%) either upon reactive loop cleavage by AspN protease or complex formation with granzyme B or caspase-1, consistent with the insertion of the cleaved reactive loop into sheet A in both types of crmA-protease complexes. NBD labels on the noninserting part of the reactive loop docking site for protease (P1 residue) or midway between the two ends of sheet A (helix F residue 101) showed no significant perturbations due to protease complexation. By contrast, labels at positions 68 and 261, lying at the end of sheet A most distal from the reactive loop, showed marked perturbations distinct from those induced by AspN cleavage and thus ascribable to granzyme B or caspase-1 proximity in the complexes. Substantial FRET between protease tryptophans and 5-dimethylaminonaphthalene-1-sulfonyl-labeled crmAs occurred in protease complexes with crmAs labeled at the 68 and 261 positions, but not the P1 position. These results suggest that granzyme B and caspase-1 are inhibited by crmA by a common mechanism involving full reactive loop insertion into sheet A and translocation of the protease to the distal end of the sheet as previously found for inhibition of other serine proteases by serpins.

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