THE CRYSTAL-STRUCTURE OF PORCINE PANCREATIC ALPHA-AMYLASE IN COMPLEX WITH THE MICROBIAL INHIBITOR TENDAMISTAT

The crystal structure of the complex formed between the 498 amino acid residue porcine pancreatic alpha-amylase (PPA) and the 74 amino acid residue inhibitor Tendamistat secreted from Streptomyces tendae, has b een determined by multiple isomorphous replacement in a crystal of spa ce group P6(5)22 (a = b = 77.7 Angstrom, c = 359.5 Angstrom). The mode l has been refined to an X-factor of 0.194 by Powell minimization appl ying strong energy constraints based on 17,964 independent reflections in the 7 to 2.5 Angstrom resolution range, and obeys standard geometr y within 0.011 Angstrom in bond lengths and 1.78 degrees in bond angle s. The final model consists of all 496 amino acid residues of PPA, 71 amino acid residues of Tendamistat (without the three N-terminal resid ues), one calcium ion, one chloride ion and 167 water molecules. PPA e xhibits the same topological fold in the complex as the uncomplexed PP A recently published by others. About 30% of the water-accessible surf ace of Tendamistat is in contact with PPA. Four segments of the polype ptide chain, with a total of 15 amino acid residues, are involved in t he binding. One segment containing the staggered Side-chains of the tr iplet Trp18, Arg19, Tyr20, typical for this class of inhibitors, binds into the catalytic site. The other segments fill out the groove in th e PPA molecule, which also binds the carbohydrate inhibitor acarbose a nd is assumed to be the substrate-binding region. This extended intera ction between Tendamistat and alpha-amylase explains the very high inh ibition constant of about 9 x 10(-12) M.