Three-dimensional structural analysis of non-pepsin-type acid proteinase using NMR and x-ray crystaliography

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Proteinase A [EC3.4.23.6] secreted by the fungus Aspergillus niger var.macrosporus is a non-pepsin-type acid proteinase. It is distinctly different from ordinary pepsin-type aspartic proteinases in primary structure, substrate specificity and reactivity to pepstatin. It remains to be elucidated which residues participate in the catalysis of the enzyme and how the mechanism operates. To make clear these queries, the three-dimensional structure of proteinase A was studied by nuclear magnetic resonance (NMR) and X-ray crystallography. The pH dependence of NMR spectra indicated that a conformational change occurs around pH 4.5 and the protein denatures irreversibly at the pH higher than 6. Distribution of cross peaks in the fingerprint region indicated that the enzyme is rich in beta-sheet and contains a tightly packed core in the molecule. Homonuclear and heteronuclear 3D-NMR spectra were measured using isotope-labeled samples. During the experiment, neither autolysis nor denaturation occurred. The spectral analysis and resonance assignments are in progress. On the other hand, the crystals of proteinase A grown at the pH around 2 gave the intensity data up to about 1.5 * resolution with the Weissenberg camera at the station BL6A2 at the Photon Factory, National Laboratory for High Energy Physics. The electron density map was obtained using the data of three isomorphous heavy atom derivatives. It showed that the protein has the molecular shape like croissant.