First of all, you don't say how long it took to first set up crystals, for them to grow, harvest, freeze and collect data on. Secondly how long did leave the peptide/substrate for your SDS PAGE experiment? If they are of a different time scale e.g. 6 hours v.s. 30 days, it may be that your enzyme is not totally dead.
Also how did you purify the alanine mutant? If you purified it on the same columns/beads as the WT protein you may have a residual amount of active protein which could cleave your peptide over the course of crystallization. You may want to use fresh beads, or treat columns with pepsin or sodium hydroxide. Not real answers I am afraid, more like suggestions.
