Single- and double-headed analogs of pyridoxamine 5′-phosphate as probes for pyridoxamine 5′-phosphate utilizing enzymes

Abstract

Single- and double-headed analogs of pyridoxamine 5′-phosphate were synthesized as cleft-depth probes for the catalytic sites of two types of enzymes, viz. those which use pyridoxamine 5′-phosphate as a substrate, i.e., pyridoxamine (pyridoxine) 5′-phosphate oxidase and those which use pyridoxamine 5′-phosphate as coenzyme, i.e., aspartate amino-transferase. These probes were prepared by borohydride reduction of the Schiff's bases formed by the interaction of pyridoxal 5′-phosphate with one or both ends of 1, n-diaminoalkanes (where n = 2, 4, 6, 8, or 12 carbons). Kinetic studies using these analogs as substrates for the oxidase revealed that the single-headed analogs exhibit values ranging from 3 K m to K m for pyridoxamine 5′-phosphate in a curvilinear relationship with chain lengths for n = 2 to 12. The short-chain substrates ( n = 2, 4) exhibit V m values greater than for pyridoxamine 5′-phosphate, whereas the long-chain ones exhibit lesser V m values. These observations may indicate the increasing hydrophobicity of the substrate with increasing chain length and thus poorer turnover. Lineweaver-Burk plots of the double-headed substrates were bilinear for the shorter chains ( n = 2, 4, 6) and linear for the longer chains ( n = 8, 12) over the concentration range of study (1 to 500 μ m). This indicates that the catalytic cleft may impede reorientation for further substrate action of the single-headed amine released from the double-headed substrate. In both cases (>50 μ m), the high K m values may reflect the ionic repulsion forces established by the different secondary amine functionalities, (CH 2) n NH 3 + for single-headed and 5′-phosphopyridoxyl for the double-headed, with active site residues thus effecting binding and ultimate turnover. These same analogs do not act as coenzymes for apo-aspartate aminotransferase or compete with pyridoxamine 5′-phosphate at the micromolar level. They do appear to bind weakly to the enzyme as observed by fluorescence quenching.