Human salivary a-amylase (HSAmy) is a component of the acquired enamel pellicle and is used by S. gordonii to colonize the oral cavity. Unlike salivary Statherin and PRPs that avidly bind to hydroxyapatite (HAp), HSAmy does not possess a highly charged N-terminus. Structural analysis of HSAmy revealed that the molecular surface of HSAmy could be divided into two opposing negatively charged potential surfaces. One such surface is centered on the substrate-binding pocket while the other, containing a cluster of twelve acidic residues, is present on the opposite side. Hypothesis: We hypothesized that the second surface might be the major hydroxyapatite-binding domain in HSAmy. Objective: To test the role of Glu76-Asp77-Glu78 tripeptide segment, part of the cluster and resembling similar acidic segments in Statherin and PRPs, in HAp binding. Methods: We mutated the Glu76-Asp77-Glu78 to Ala76-Ala77-Ala78 (HSAmy-N) using HSAmy cDNA by PCR. HSAmy-N was expressed in a baculovirus system and purified using ion exchange and gel filtration techniques. The HAp binding abilities of HSAmy-N and HSAmy were compared using a HAp binding assay. Results: 1) Removal of the successive acidic tripeptide segment resulted in the elution of HSAmy-N in the flow-through indicating that it does not bind to the DE52 column matrix; and 2) HAp binding assay revealed that 11% of HSAmy was unbound compared to 26% for HSAmy-N. FTIR spectroscopy and western blot analysis of the bound fraction confirmed the weakened ability of HSAmy-N in HAp binding. Conclusion: These results support the hypothesis that the second negative electrostatic potential surface opposite to the substrate binding pocket might be a major domain in the binding of HSAmy to HAp surfaces. This work was supported by USPHS Grant DE12585.

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