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The crystal structure of 0.19 alpha-amylase inhibitor (0.19 AI) from wheat kernel was determined by the multiple-isomorphous replacement method coupled with density modification and noncrystallographic symmetry averaging and then refined by simulated annealing using diffraction data to 2.06 A resolution (R = 18.7%, free R = 22.3%). The asymmetric unit has four molecules of 0.19 AI, each comprised of 124 amino acid residues. Electron density for residues 1-4 and 69-77 is absent in all subunits, probably because of the intrinsic flexibility of these segments. Each subunit has four major alpha-helices and one one-turn helix which are arranged in the up-and-down manner, maintaining the favorable packing modes of the alpha-helices. 0.19 AI, however, has two short antiparallel beta-strands. All 10 cysteine residues in 0.19 AI form disulfide bonds (C6-C52, C20-C41, C28-C83, C42-C99, and C54-C115), consistent with the assignments made biochemically for 0.28 AI from wheat kernel and by NMR analysis of the bifunctional alpha-amylase/trypsin inhibitor from ragi seeds (RBI). The disulfide bond patterns in these AIs are similar to those in the hydrophobic protein from soybean (HPS), which lack only the bond corresponding to C28-C83 in 0.19 AI. Extensive interactions occurred between particular pairs of 0.19 AI subunits, mainly involving hydrophobic residues. Comparisons of the structures of 0.19 AI, RBI, and HPS showed that the arrangements of the major alpha-helices are similar but the conformations of the remaining residues differ markedly. The present X-ray analysis for 0.19 AI and the NMR analysis for RBI suggest that all the AIs in this family have a common fold. The alpha-amylase binding site is discussed on the basis of the tertiary and quaternary structures of 0.19 AI together with biochemical and spectroscopic data for AIs.

In the presence of ammonium sulfate the absorption spectra of a peroxidase from the fungus Arthromyces ramosus (ARP) showed that the low-spin component increased as the pH increased from 6.0 to 9.0, whereas in its absence ARP remained in the high-spin state in the pH range investigated. The crystal structure of ARP at pH 4.5 in the presence of ammonium sulfate at 1.8 A resolution showed that the electron density at the 6th position of the heine iron seen at pH 7.5 had disappeared and that the iron atom deviated markedly from the heme plane. These observations strongly suggest that under physiological conditions the heine of ARP is in the pentacoordinated high-spin state and that at a high pH the heine iron is able to bind ammonia, forming the low-spin state. The location of the water molecule at the distal side of the heme in peroxidases is also discussed.

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