The crystal structure of HPTP-B, a human isoenzyme of the low molecular weight phosphotyrosyl phosphatase (LMW PTPase) is reported here at a resolution of 1.6 Å . This high resolution structure of the second human LMW PTPase isoenzyme provides the opportunity to examine the structural basis of different substrate and inhibitor/activator responses. The crystal packing of HPTP-B positions a normally surface-exposed arginine in a position equivalent to the tyrosyl substrate. A comparison of all deposited crystallographic coordinates of these PTPases reveals three atomic positions within the active site cavity occupied by hydrogen bond donor or acceptor atoms on bound molecules, suggesting useful design elements for synthetic inhibitors. A selection of inhibitor and activator molecules as well as small molecule and peptide substrates were tested against each human isoenzyme. These results along with the crystal packing seen in HPTP-B suggest relevant sequence elements in the currently unknown target sequence.Tyrosine phosphorylation and dephosphorylation are critical components of eukaryotic signaling. The superfamily of protein-tyrosine phosphatases (PTPases) 2 is defined in part by a canonical CX 5 R(S/T) active site motif (P-loop), and can be divided into families based on substrate specificity and protein size (1-3). Following the classification scheme of Alonso et al. (1), the receptor-like PTPases such as CD45 (4) and non-receptor PTPases such as PTP1B (5) are subdivisions of the class I Cys-based family, multidomain proteins where the canonical phosphatase domain is roughly 30 kDa. The low molecular weight (LMW) PTPase, a soluble 18-kDa protein with essentially no sequence similarity to the other families beyond the active site P-loop (6), is the only member of the class II Cys-based family. Two isoenzyme forms of the LMW PTPase are expressed in humans, HPTP-A and HPTP-B, identical in sequence except for an mRNA splice variant sequence corresponding to residues 40 -73 (7).Although all PTPases are thought to have the same basic mechanism, the families show marked differences in substrate specificity and small molecule modulation. Within the class II family those differences can be sorted into either an HPTP-A or HPTP-B type response. Both isoenzyme forms have been isolated from rat, chicken, Xenopus laevis, and Drosophila (8 -13), whereas the single Tritrichomonas fetus enzyme is similar to HPTP-A (14) and the Mycobacterium tuberculosis, yeast, and bovine enzymes are similar to HPTP-B (15-18). The human isoenzymes exhibit a dramatically different response to a variety of purine molecules (19,20). In particular, adenine has been shown to inhibit HPTP-A and activate HPTP-B, whereas hypoxanthine activates HPTP-A with little to no effect on HPTP-B. It has also been reported that the rat isoenzyme ACP2 is activated 900% by cGMP, whereas the activity of isoenzyme ACP1 is unchanged (21). Adenine has been co-crystallized with the Saccharomyces cerevisiae LMW PTPase, and the structure suggests a basis for . The adeni...

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