Alkaline Phosphatase Structure Influences Heat Stability – Joseph LHuillier and Adam Miller

Alkaline phosphatase (AP) enzyme structure is highly conserved among phyla, but differences in AP heat stability exist. Human placental AP (HPLAP) contains a heat-stable amino acid 429, a hydrophobic pocket, and an RY cluster– all likely involved in AP stability. We hypothesize that HPLAP will be most heat stable compared to bovine intestinal AP (BIAP) which will be least heat stable due to the absence of these structures. Human intestinal AP (IAP) is expected to have an intermediate level of heat stability due to the presence of a hydrophobic pocket only. Heat stability was determined by measuring enzyme reaction velocity with substrate p-nitrophenol phosphate run at 37°C and 55°C. Both BIAP and IAP showed similar decrease in reaction velocity at 55°C, while HPLAP maintained high reactivity at 55°C no different that 37°C. These results indicate that the hydrophobic pocket with AA429 may not be sufficient for heat stability, but that the hydrophobic pocket may be necessary. The results will help elucidate the relative importance of AP structural components in heat stability. Poster