My worst thoughts, then, were confirmed.-Edgar Allan Poe (1843), The Pit and the Pendulum

ABSTRACT:Raloxifene and 4-hydroxytamoxifen (4-OHT) are important estrogen-related drugs used in the treatment of osteoporosis and breast cancer. Sulfation is involved in the metabolism and inactivation of both compounds in human tissues, although the sulfotransferase (SULT) isoforms involved in their conjugation have not been well described. The ability of seven expressed SULT isoforms to sulfate raloxifene and 4-OHT was investigated. Raloxifene was conjugated by all seven SULT isoforms tested, whereas 4-OHT was conjugated only by SULTs 1A1, 1E1, and 2A1. Characterization of raloxifene and 4-OHT sulfation demonstrates that sulfation can occur at therapeutic concentrations. SULT1E1 displayed the lowest K m (0.2 M) for 4-OHT sulfation and SULT2A1 the lowest (0.3 M) for raloxifene sulfation. SULT1E1 was the only isoform exhibiting detectable levels of raloxifene disulfation activity. Modeling of the interactions of raloxifene in the active site of SULT1E1 indicates that both hydroxyl groups of raloxifene can be readily positioned in proximity to the sulfonyl group of 3-phosphoadenosine 5-phosphosulfate and the catalytically important His107 residue. Both raloxifene and 4-OHT sulfation activities were detectable in all human liver cytosols tested. 4-OHT sulfation was detected in cytosol prepared from endometrial biopsies of normal women obtained during the proliferative and secretory phases of the same menstrual cycle. In contrast, raloxifene sulfation was detectable only in secretory phase cytosols in association with SULT1E1 activity. In summary, several human SULT isoforms are capable of sulfating raloxifene and 4-OHT. Tissue-specific expression of the individual SULT isoforms may have important roles in the regulation of the activity of these compounds.

Phosphomevalonate kinase catalyzes an essential step in the so-called mevalonate pathway, which appears to be the sole pathway for the biosynthesis of sterols and other isoprenoids in mammals and archea. Despite the well documented importance of this pathway in the cause and prevention of human disease and that it is the biosynthetic root of an enormous diverse class of metabolites, the mechanism of phosphomevalonate kinase from any organism is not yet well characterized. The first structure of a phosphomevalonate kinase from Streptococcus pneumoniae was solved recently. The enzyme exhibits an atypical P-loop that is a conserved defining feature of the GHMP kinase superfamily. In this study, the kinetic mechanism of the S. pneumoniae enzyme is characterized in the forward and reverse directions using a combination of classical initial-rate methods including alternate substrate inhibition using ADPbetaS. The inhibition patterns strongly support that in either direction the substrates bind randomly to the enzyme prior to chemistry, a random sequential bi-bi mechanism. The kinetic constants are as follows: k(cat(forward)) = 3.4 s(-1), K(i(ATP)) = 137 microm, K(m(ATP)) = 74 microm, K(i(pmev)) = 7.7 microm, K(m(pmev)) = 4.2 microm; k(cat(reverse)) = 3.9 s(-1), K(i(ADP)) = 410 microm, K(m(ADP)) = 350 microm, K(i(ppmev)) = 14 microm, K(m(ppmev)) = 12 microm, where pmev and ppmev represent phosphomevalonate and diphosphomevalonate, respectively.

The atomic force microscope (AFM) measures force and displacement with high sensitivity and submillisecond temporal resolution. By functionalizing the AFM probe with specific chemical groups or macromolecules it is possible to characterize the chemical and physical properties of single molecules on the nanometer scale. In this paper we discuss the key issues that must be addressed when designing and characterizing a successful immobilization chemistry, and describe the chemistry we developed to covalently immobilize oligonucleotides in a specific orientation.

ATP sulfurylase catalyzes the first step in the activation of sulfate by transferring the adenylyl-moiety (AMP approximately ) of ATP to sulfate to form adenosine 5'-phosphosulfate (APS) and pyrophosphate (PP(i)). Subsequently, APS kinase mediates transfer of the gamma-phosphoryl group of ATP to APS to form 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and ADP. The recently determined crystal structure of yeast ATP sulfurylase suggests that its C-terminal domain is structurally quite independent from the other domains, and not essential for catalytic activity. It seems, however, to dictate the oligomerization state of the protein. Here we show that truncation of this domain results in a monomeric enzyme with slightly enhanced catalytic efficiency. Structural alignment of the C-terminal domain indicated that it is extremely similar in its fold to APS kinase although not catalytically competent. While carrying out these structural and functional studies a surface groove was noted. Careful inspection and modeling revealed that the groove is sufficiently deep and wide, as well as properly positioned, to act as a substrate channel between the ATP sulfurylase and APS kinase-like domains of the enzyme.