STRUCTURAL REPLACEMENT OF ACTIVE-SITE MONOVALENT CATIONS BY THE EPSILON-AMINO GROUP OF LYSINE IN THE ATPASE FRAGMENT OF BOVINE HSC70

We have assessed the ability of the epsilon-amino group of a non-nativ e lysine chain to substitute for a monovalent cation in an enzyme acti ve site. In the bovine Hsc70 ATPase fragment, mutation of cysteine 17 or aspartic acid 206 to lysine potentially allows the replacement of a n active site potassium ion with the epsilon-amino nitrogen. We examin ed the ATP hydrolysis kinetics and crystal structures of isolated muta nt ATPase domains. The introduced epsilon-amino nitrogen in the C17K m utant occupies a significantly different position than the potassium i on. The introduced epsilon-amino nitrogen in the D206K mutant occupies a position indistinguishable from that of the potassium in the wild-t ype structure. Each mutant retains <5% ATPase activity when compared t o the wild type under physiological conditions (potassium buffer) alth ough substrate binding is tighter, probably as a consequence of slower release. It is possible to construct a very good structural mimic of bound cation which suffices for substrate binding but not for catalyti c activity.