The roles of three conserved active site carboxylic acids (D197, E233, and D300) in the catalytic mechanism of human pancreatic alpha-amylase (HPA) were studied by utilizing site-directed mutagenesis in combination with structural and kinetic analyses of the resultant enzymes. All three residues were mutated to both alanine and the respective amide, and a double alanine mutant (E233A/D300A) was also generated. Structural analyses demonstrated that there were no significant differences in global fold for the mutant enzymes. Kinetic analyses were performed on the mutants, utilizing a range of substrates. All results suggested that D197 was the nucleophile, as virtually all activity (>10(5)-fold decrease in k(cat) values) was lost for the enzymes mutated at this position when assayed with several substrates. The significantly greater second-order rate constant of E233 mutants on "activated" substrates (k(cat)/K(m) value for alpha-maltotriosyl fluoride = 15 s(-)(1) mM(-)(1)) compared with "unactivated" substrates (k(cat)/K(m) value for maltopentaose = 0.0030 s(-)(1) mM(-)(1)) strongly suggested that E233 is the general acid catalyst, as did the pH-activity profiles. Transglycosylation was favored over hydrolysis for the reactions of several of the enzymes mutated at D300. At the least, this suggests an overall impairment of the catalytic mechanism where the reaction then proceeds using the better acceptor (oligosaccharide instead of water). This may also suggest that D300 plays a crucial role in enzymic interactions with the nucleophilic water during the hydrolysis of the glycosidic bond.

A mechanistic study of the poorly understood pathway by which the inhibitor acarbose is enzymatically rearranged by human pancreatic alpha-amylase has been conducted by structurally examining the binding modes of the related inhibitors isoacarbose and acarviosine-glucose, and by novel kinetic measurements of all three inhibitors under conditions that demonstrate this rearrangement process. Unlike acarbose, isoacarbose has a unique terminal alpha-(1-6) linkage to glucose and is found to be resistant to enzymatic rearrangement. This terminal glucose unit is found to bind in the +3 subsite and for the first time reveals the interactions that occur in this part of the active site cleft with certainty. These results also suggest that the +3 binding subsite may be sufficiently flexible to bind the alpha-(1-6) branch points in polysaccharide substrates, and therefore may play a role in allowing efficient cleavage in the direct vicinity of such junctures. Also found to be resistant to enzymatic rearrangement was acarviosine-glucose, which has one fewer glucose unit than acarbose. Collectively, structural studies of all three inhibitors and the specific cleavage pattern of HPA make it possible to outline the simplest sequence of enzymatic reactions likely involved upon acarbose binding. Prominent features incorporated into the starting structure of acarbose to facilitate the synthesis of the final tightly bound pseudo-pentasaccharide product are the restricted availability of hydrolyzable bonds and the placement of the transition state-like acarviosine group. Additional "in situ" experiments designed to elongate and thereby optimize isoacarbose and acarviosine-glucose inhibition using the activated substrate alphaG3F demonstrate the feasibility of this approach and that the principles outlined for acarbose rearrangement can be used to predict the final products that were obtained.

The increasing prevalence of diabetes has accelerated the search for new drugs derived from natural sources. To define the functional features of two such families of compounds, the flavonols and the ethyl caffeates, we have determined the high-resolution structures of representative inhibitors in complex with human pancreatic α-amylase. Myricetin binds at the active site and interacts directly with the catalytic residues despite its bulky planar nature. Notably, it reduces the normal conformational flexibility of the adjacent substrate binding cleft. In contrast, bound ethyl caffeate acts by disordering precisely those polypeptide chain segments that make up the active site binding cleft. It also operates from binding sites far removed from the active site, a property not observed in any other class of human α-amylase inhibitor studied to date. Given the current inadequacy of drugs directed at diabetes, the use of optimized flavonols and ethyl caffeates may present an alternative therapeutic route.

Colicins kill E. coli by a process that involves binding to a surface receptor, entering the cell, and, finally, intoxicating it. The lethal action of colicin E3 is a specific cleavage in the ribosomal decoding A site. The crystal structure of colicin E3, reported here in a binary complex with its immunity protein (IP), reveals a Y-shaped molecule with the receptor binding domain forming a 100 A long stalk and the two globular heads of the translocation domain (T) and the catalytic domain (C) comprising the two arms. Active site residues are D510, H513, E517, and R545. IP is buried between T and C. Rather than blocking the active site, IP prevents access of the active site to the ribosome.