Spontaneous mutations in the human LIS1 gene are responsible for Type I lissencephaly ("smooth brain"). The distribution of neurons within the cerebral cortex of lissencephalic children appears randomized, probably owing to a defect in neuronal migration during early development. LIS1 has been implicated in the dynein pathway by genetic analyses in fungi. We previously reported that the vertebrate LIS1 co-localized with dynein at prometaphase kinetochores, and interference with LIS1 function at kinetochore caused misalignment of chromosomes onto the metaphase plate. This leads to a hypothesis that LIS1 might regulate kinetochore protein targeting. In order to test this hypothesis, I created dominant inhibitory constructs of LIS1. After removal of the endogenous LIS1 from the kinetochore by overexpression of the N-terminal self-association domain of LIS1, dynein and dynactin remained at the kinetochores. This result indicated that LIS1 is not required for dynein to localize at the kinetochore. Next, CLIP-170 was displaced from the kinetochores in the LIS1 full-length and the C-terminal WD-repeat overexpressers, suggesting a role for LIS1 in targeting CLIP-170 onto kinetochores. LIS1 was co-immunoprecipitated with dynein and dynactin. Its association with kinetochores was mediated by dynein and dynactin, suggesting LIS1 might interact directly with subunits of dynein and/or dynactin complexes. I found that LIS1 interacted with the heavy and intermediate chains (HC and IC) of dynein complex, and the dynamitin subunit of dynactin complex. In addition to kinetochore targeting, the LIS1 C-terminal WD-repeat domain was responsible for interactions with dynein and dynactin. Interestingly, LIS 1 interacted with two distinct sites on HC: one in the stem region containing the subunit-binding domain, and the other in the first AAA motif of the motor domain, which is indispensable for the ATPase function of the motor protein. This LIS1-dynein motor domain interaction suggests a role for LIS1 in regulating dynein motor activity. To test this hypothesis, changes of dynein ATPase activity was measured in the presence of LIS1 protein. The ATPase activity of dynein was stimulated by the addition of a recombinant LIS1 protein. Besides kinetochores, others and we have found LIS1 also localized at microtubule plus ends. LIS1 may mediate dynein and dynactin mitotic functions at these ends by interacting with astral microtubules at cortex, and associating with the spindle microtubules at kinetochores. Overexpression of LIS1 displaced dynein and dynactin from the microtubule plus ends, and mitotic progression was severely perturbed in LIS1 overexpressers. These results suggested that the role for LIS1 at microtubule plus ends is to regulate dynein and dynactin interactions with various subcellular structures. Results from my thesis research clearly favored the conclusion that LIS1 activates dynein ATPase activity through its interaction with the motor domain, and this activation is important to establish an interaction between dynein and microtubule plus ends during mitosis. I believe that my thesis work not only has provided ample implications regarding dynein dysfunction in disease formation, but also has laid a significant groundwork for more future studies in regulations of the increasing array of dynein functions.

Proliferation and differentiation are essential processes for the growth and development of higher eukaryotic organisms. Regulation of gene expression is essential for control of cell division and differentiation. Normal eukaryotic cells have a limited proliferative capacity, and ultimately undergo cellular senescence and apoptosis. Terminal differentiation of cells is associated with loss of proliferative capacity and acquisition of specialized functions. Proliferation and differentiation are processes required for the creation and maintenance of diverse tissues both during embryonic development and postnatal life. The cell cycle is the process by which cells reproduce, and requires duplication and segregation of hereditary material. Loss of cell cycle control leads to genetic instability and cancer. Expression of replication-dependent histone genes is tightly coupled to DNA synthesis, thus making histone genes a good model for studying cell cycle regulation. The HiNF-D complex interacts with all five classes (H1, H2A, H2B, H3 and H4) of histone genes in a cell cycle-dependent manner. The CCAAT displacement protein (CDP)/Cux and the tumor suppressor pRB are key components of the HiNF-D complex. However, the molecular interactions that enable CDP/Cux and pRB to form a complex and thus convey cell growth regulatory information onto histone gene promoters are poorly understood. Transient transfection assays show that CDP/Cux represses the histone H4 promoter and that the pRB large pocket domain functions with CDP/Cux as a co-repressor. Direct interaction between CDP/Cux C-terminus and the pRB pocket domain was observed in GST pull-down assays. Furthermore, co-immunoprecipitation assays and immunofluorescence microscopy established that CDP/Cux and pRB form complexes in vivo and associate in situ. pRB interaction and co-repression with CDP/Cux is independent of pRB phosphosphorylation sites, as revealed by GST pull-down assays and transient transfection assays using a series of pRB mutant proteins. Thus, several converging lines of evidence indicate that complexes between CDP/Cux and pRB repress cell cycle-regulated histone gene promoters. CDP/Cux is regulated by phosphorylation and acetylation at the C-terminus, which contains two repressor domains and interacts with histone deacetylase HDAC1. In vivo function of the CDP/Cux C-terminus in development and gene regulation was assessed in genetically targeted mice (Cutl1tm2Ejn, referred to as Cutl1ΔC). The mice express a mutant CDP/Cux protein with a deletion of the C-terminus including the homeodomain. Indirect immunofluorescence microscopy showed that the mutant protein exhibited significantly reduced nuclear localization in comparison to the wildtype protein. Consistent with these data, DNA binding activity of HiNF-D was lost in nuclear extracts derived from mouse embryonic fibroblasts (MEFs) or adult tissues of homozygous mutant (Cutl1 ΔC -/-) mice, indicating the functional loss of CDP/Cux in the nucleus. No significant difference in growth characteristics or total histone H4 mRNA levels was observed between wildtype and Cutl1 ΔC -/- MEFs in culture. However, the histone H4.1 (murine FO108) gene containing CDP/Cux binding sites have reduced expression levels in homozygous mutant MEFs. Stringent control of growth and differentiation appears to be compromised in vivo. Homozygous mutant mice exhibit stunted growth (20-50% weight reduction), a high postnatal death rate of 60-70%, sparse abnormal coat hair and severely reduced fertility. Hair follicle deformities and severely diminished fertility in Cutl1 ΔC -/- mice suggest that CDP/Cux is required for normal development of dermal tissues and reproductive functions. Together the data presented in this dissertation provide new insight into the in vivo functions of CDP/Cux in the regulation of histone gene expression, growth control and differentiation.

Summary Basic biological processes require gene expression. Tightly regulated molecules known as transcription factors mediate the expression of genes in development and disease. Signal transduction pathways, which respond to environmental cues or stressors are major regulators of the transcription factors. Use of macromolecular synthesis inhibitors in models of normal neurodevelopment and neurodegenerative cell death has led to the discovery that gene expression is required for these processes to occur (Martin et. al.,(1988), J Cell Biol 106p829). To date, however, the identities of very few of the genes required in these events have been revealed. Hence, the activation or requirement of specific signaling pathways leading to the expression of known apoptotic genes is not well established. Utilizing the neurothrophic factor deprivation and neurotoxin models of programmed cell death we address these gaps in our understanding of the molecular mechanism of apoptosis as it occurs in neuronal cell death. Nerve growth factor (NGF) withdrawal from PC12 cells leads to the activation of p38 and apoptosis. The functional significance of 38 activation in this paradigm of cell death is not known. To increase our understanding of apoptosis I examined the requirement for p38 activity in pro-apoptotic gene expression in PC12 cells. I performed a subtractive hybridization that led to the identification of the monoamine oxidase (MAG) gene as induced in response to NGF withdrawal. Using the p38 inhibitor PD169316 I showed that the NGF withdrawal stimulated induction of the MAG gene and apoptosis is blocked by inhibition of the p38 MAP kinase pathway. I also determined that the MAG inhibitor clorgyline blocked cell death indicating that MAG activity contributes to the cell death caused by NGF withdrawal. Together, these data indicate that the p38 MAP kinase pathway targets the MAG gene in response to apoptotic stimuli. To study the requirement for the JNK signaling pathway in neurodegeneration I stimulated primary cortical neurons with the neurotoxin arsenite. Arsenite treatment of primary neurons leads to both JNK and p38 activation and subsequently apoptosis. Utilizing transgenic mice lacking the JNK3 gene I demonstrated that JNK3 specifically contributes to the effects of arsenite in these cells. Ribonuclease protection assays were used to identify Fas ligand as a molecule whose arsenite-induced expression is dependent on the JNK3 signal transduction pathway. Furthermore, I have shown that neurons deficient in signaling mediated by the receptor for Fas ligand are resistant to cell death due to arsenite treatment. These results in total have established that the JNK3 mediated expression of Fas ligand contributes to the arsenite induced death of cortical neurons. In summary, the work presented in these studies identifies the JNK and p38 MAP kinase signal transduction pathways as mediators of apoptosis in neuronal cells. Importantly, I have provided evidence that these stress activated pathways are responsible for the expression of specific genes in apoptotic neuronal cells.

KCNE β-subunits modulate KCNQ1 (Q1) voltage-gate K+channels providing the current diversity required for Q1 channels to function in a wide variety of cell types and tissues. In the present thesis, the stoichiometry of KCNE1 (E1) β-subunits in functioning Q1 channels is investigated, along with the formation of heteromeric channel complexes, complexes containing 2 different KCNE β-subunits. The chemical approaches used to answer these questions were then expanded to generate a novel labeling reagent. To determine the stoichiometry of the Q1/E1 complex, I devised an iterative subunit counting approach that relies on a chemically releasable K+channel blocking reagent. The extracellularly applied reagent irreversibly blocks charybdotoxin (CTX) sensitive Q1 channels by chemically modifying E1 peptides that contain an N-terminal cysteine residue. Chemical release of the inhibitor and subsequent iterative applications of the reagent reported that Q1 channels partner with two KCNE β-subunits. To determine whether heteromeric Q1-KCNE complexes form, I synthesized a similar, but non-cleavable, K+channel blocking reagent that detects specific KCNE peptides in functioning complexes by irreversible channel inhibition. Using this “KCNE sensor”, heteromeric Q1/E1/E3, Q1/E1/E4 and Q1/E3/E4 complexes were shown to form, traffic to the cell surface and function. Using mathematical subtraction to visualize the irreversibly blocked current, the currents and gating kinetics of the different heteromeric complexes were revealed and a hierarchy of KCNE subunit modulation of Q1 channels was determined: E3>E1>>E4. Building on this technology, a chemically releasable K+ channel blocking reagent was created to specifically label KCNE β-subunits with biotin. The reagent delivers biotin to CTX sensitive Q1 channels and labeling occurs through free thiols provided by either cysteine residues or thiol modified sugars. This preliminary data demonstrates a novel strategy for labeling endogenous K+ channels in native cells.

Human immunodeficiency virus type 1 (HIV-1) infections have different patterns of expression in different T-cell lines. HIV-1 encodes regulatory as well as structural genes. The role of HIV-1 regulatory gene expression in determining different patterns of infection was explored in four T-cell lines: C8166, H9, A3.01, and Jurkat. The hypothesis being tested was that differences in the expression of regulatory genes would determine differences in the kinetics of infection. To study patterns of regulatory and structural gene expression, RNA was isolated from cultures infected with HIV-1-NL4-3 (NL4-3). During the early and acute phases of infection, the absolute amounts of viral RNA differed in the four T-cell lines. However, the relative proportions of messages for regulatory and structural genes were similar. Thus, differences in the kinetics of infection in C8166, H9, A3.01 and Jurkat cells were not determined by differences in the relative levels of expression of regulatory and structural genes. Analyses of RNA samples from the chronic phase of infection revealed the consistent appearance of novel RNase sensitive sites in H9 and Jurkat cultures. These marked the emergence of viral variants with high ability to establish chronic virus producers. These variants were specifically selected in the chronic phase since they did not undergo selection during serial passage of the virus through the lytic phase of infection. Sequence analysis of the region with the novel RNase sensitive sites revealed the co-mapping of nucleotide changes with each of the novel sites. Most of these differences represented a sense mutation in tat and the abrogation of the initiator methionine of vpu. However, the selected mutations in tat and vpuwere not sufficient, by themselves, to affect the ability of NL4-3 to establish chronic virus producers (Chapters I and II). Further studies on the roles of viral sequences in the chronic phase of infection were undertaken using constructed viruses. Two molecularly cloned viruses, NL4-3 and HIV-1-HXB-2 (HXB-2), were used as parents. NL4-3 has a low ability to establish chronic virus producers. In contrast, HXB-2 has a high ability to establish chronic virus producers. NL4-3 encodes all known HIV-1 genes, whereas HXB-2 is defective for three auxiliary genes: vpr, vpu, and nef. In addition, both viruses differ at other positions throughout the genome. The first series of constructed viruses tested whether differences in auxiliary gene expression determined differences in the ability of NL4-3 and HXB-2 to establish chronic virus producers. NL4-3 mutants containing all possible combinations of the three defective genes in HXB-2 were constructed. Analysis of the ability of these mutants to establish chronic virus producers revealed that vpr and neflimit the ability of NL4-3 to establish chronic virus producers. This was shown by viruses with defects in both of these genes having high ability to establish chronic virus producers (Chapter III). The second series of constructs tested for the roles of non-auxiliary as well as auxiliary gene sequences on chronic virus production by creating recombinants between NL4-3 and HXB-2. Tests of these recombinants revealed that a gag, pol, vif, and vpr fragment could affect the ability of fragments containing defective auxiliary genes to establish chronic virus producers. Taken together, these results indicate that vpr, nef, and 5' internal sequences play important roles in determining the ability to establish chronic virus producers (Chapter IV).

The promoter sequence of the mouse high affinity neurotensin receptor, Ntr-1, gene was cloned and characterized, sequences required for positive regulation in N1E-115 cells were localized, and at least two different peptides from these cells were shown to make specific contacts within the most potent positive regulatory element. A mouse neuroblastoma cell line, N1E-115, treated with 1.5% DMSO for 72 hours induces gene expression of both endogenous Ntr-l, and reporter constructs driven by the NTR-1 promoter, by 3 - 4 fold. The sequence ofthe NTR-1 promoter has no canonical TATA box, but is GC rich and contains consensus SP1, CACCC, CRE, and initiator elements. These elements are located within a 193 base positive regulatory region required for DMSO responsive activity and contains the transcriptional start site. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC rich palindrome are crucial cis-regulatory elements for DMSO induction. The SP1 element, an NGFI-A-related element, and the 5' end of the positive regulatory region are required for maintaining basal expression in N1E-115 cells. Cell type differences in the cis-regulatory elements that mediate both DMSO induction and maintenance of basal expression are observed. Characterization of proteins in N1E-115 cells that make specific contacts within the CACCC element identified at least two peptides with predicted sizes of 57 kd and 97 kd. Two dimensional UV crosslinking indicates that these proteins might contribute to inducible gel shift complexes that require the CACCC element. Several previously characterized CACCC binding proteins, belonging to the Kruppel-like family of transcription factors, were tested by supershift analysis for their ability to contribute to NTR-1 CACCC complexes. In fact, a protein closely related to SP1 does bind the CACCC element in N1E-115 cells, but of the other Kruppel-like protein tested, only BKLF contributes to a minor complex in N1E-115 cells. These results provide evidence for the complex regulation of Ntr-1 gene expression mediated by the cooperation of several cis-regulatory elements including a CACCC Kruppel-like binding element.

CP1 (encoded by the gene CEP1) is a sequence-specific DNA-binding protein of Saccharomyces cerevisiae that recognizes a sequence element (CDEI) found in both yeast centromeres and gene promoters. Strains lacking CP1 are viable but exhibit defects in growth, chromosome segregation, and methionine biosynthesis. To investigate the basis of the methionine requirement, a YEp24-based yeast genomic DNA library was screened for plasmids which suppressed the methionine auxotrophy of a cep1 null mutant. The suppressing plasmids contained either CEP1 or DNA derived from the PHO4 locus. PHO4 encodes a factor which positively regulates transcription of genes involved in phosphate metabolism via an interaction with CDEI-like elements within the promoters of these genes. Subcloning experiments confirmed that suppression correlated with increased dosage of PHO4. PHO4c, pho80, and pho84 mutations, all of which lead to constitutive activation of the PHO4 transcription factor, also suppressed cep1 methionine auxotrophy. The suppression appeared to be a direct effect of PHO4, not a secondary effect of PHO regulon derepression, and was dependent on a second transcriptional regulatory protein encoded by PHO2. Spontaneously arising extragenic suppressors of the cep1 methionine auxotrophy were also isolated; approximately one-third of the them were alleles of pho80. While PHO4 overexpression suppressed the methionine auxotrophy of a cep1 mutant, CEP1 overexpression failed to suppress the phenotype of a pho4 mutant; however, a cep1 null mutation suppressed the low-Pi growth deficiency of a pho84 mutant. The results suggest that CP1 functions as a transcriptional regulator of MET genes, and that activation of PHO4 restores expression to those genes transcriptionally-disabled by the cep1mutation. The results also suggest the existence of a network that cross-regulates transcription of genes involved in methionine biosynthesis and phosphate metabolism. A direct molecular approach to investigate CP1's role in MET gene expression was also taken. CDEI sites are associated with the promoter regions of most MET genes, but only MET16, the gene encoding PAPS reductase, has been shown to require CP1 for expression; both PAPS reductase activity, and MET16 mRNA are absent in cep1 mutants. Results of the present study demonstrate that CP1 participates in two systems which regulate expression of MET16, one triggered by methionine starvation and requiring the transactivator MET4 (pathway-specific control), and the other triggered by starvation for many different amino acids and requiring GCN4 (general control). CP1 was shown to mediate its regulatory function through the upstream CDEI site, and to act directly or indirectly to modulate the chromatin structure of the MET16 promoter. In addition, the pho80 mutation was found to partially restore MET16 expression to the cep1 strain, confirming the proposed nature of PHO4 suppression. A second methionine biosynthetic gene MET25, was also analyzed. Like MET16, MET25 was found to be regulated by both pathway-specific and general control mechanisms, but in contrast to MET16, CP1 only participated in the pathway-specific response of this gene. The results demonstrate that CP1, possibly by modulating changes in chromatin structure, assists the regulatory proteins MET4 and GCN4 in activating transcription of MET genes.

Expression of the cell cycle dependent FO10S human H4 histone gene is regulated at both the transcriptional and post-transcriptional levels. We have investigated the 5' promoter elements mediating the transcriptional aspects of its regulation. A detailed in vivo and in vitro transcriptional analysis of promoter deletion mutants from this gene has identified three positive regulatory elements and two potentially negative regulatory elements within the first 1000 base pairs upstream of the transcription initiation site. In addition, the minimal promoter located within the first 70 base pairs is required for accurate transcription initiation and contains one of two in vivo identified protein-DNA interactions, site II. Binding of the nuclear factor HiNF-D to this region was correlated with the turn-on of histone gene transcription following stimulation of quiescent normal diploid fibroblasts to re-enter the proliferative phase. The most proximal positive regulatory element contains the other in vivo identified protein-DNA interaction, site I. Results from a series of in vitroprotein-DNA interaction studies revealed the binding of two nuclear factors to this element. One protein, HiNF-C, is indistinguishable from the transcription factor Sp1 while the other, HiNF-E, is a novel, potentially histone-specific member of the ATF transcription factor family. Binding of HiNF-C was required to stabilize the interaction of HiNF-E and together this region stimulated transcription 5 fold. The near-distal transcription activator region lies between -418 and -213 base pairs and forms a single protein- DNA complex, H4UA-1. The interaction domain for H4UA-1 contains recognition sequences for both the thyroid hormone receptor and the nuclear factor CTF/NF-1. The far-distal activator region (-730 and -589 base pairs) was the strongest positive regulatory element identified in the H4 promoter. This region increased transcription 10 fold and contains three protein-DNA interactions. One of the factors, H4UA-2, is an ATF transcription factor closely related to the HiNF-E interaction in the proximal positive element. These studies have defined the functional human H4 histone promoter to be a complex, modular structure extending at least 1000 base pairs.

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicles are targeted to sites of exocytosis on the plasma membrane in part by a conserved multi-subunit protein complex termed the exocyst. In addition to tethering vesicles to the plasma membrane, the exocyst complex and components therein may also add a layer of regulation by directly controlling assembly of the SNARE complex, which is required for membrane fusion, as well as other regulatory factors such as Sec1p. In the past, we have shown that Sec6p interacts with Sec9p in vivo and that that interaction retards binary SNARE complex formation in a SNARE assembly assay. Though many interactions have been mapped using in vitro methods, confirming them in vivoand placing them into the context of a complete model that accounts for all observed interactions (and lack of interactions) has proven difficult. In order to address these problems, I have studied the interactions between Sec6p and other factors involved in exocytosis at the plasma membrane via in vivo methods. My hypothesis was that Sec6p interaction with Sec9p and subsequent inhibition of SNARE complex assembly in vitro was an intermediate state and Sec6p was part of a set of cofactors that accelerated SNARE complex assembly in vivo. To test this hypothesis I showed that the interaction between the plasma membrane t-SNARE Sec9p and the yeast exocyst subunit Sec6p can be observed in vivoand designed point mutations to disrupt that interaction. Interestingly, I also showed that Sec6p:Sec9p interaction involves the free pool of Sec6p rather than the exocyst bound fraction of Sec6p. Point mutations in the N-terminal domain of Sec6p result in temperature sensitive growth and secretion defects, without loss of Sec6p-Sec9p interaction. However, at the non-permissive temperature, the exocyst subunits Sec5p, Sec10p and Sec15p are mislocalized and are absent from the exocyst complex. The resulting subcomplex, containing Sec3p, Sec8p, Exo70p and Exo84p, remains stably assembled and localized at sites of polarized secretion. This subcomplex is likely due to disruption of interaction between Sec6p and Sec5p, and may be similar to that observed at restrictive temperatures in the sec6-54temperature sensitive mutant. Additionally, one of the sec6 temperature sensitive mutants displays a loss of binding to the yeast regulatory protein Sec1p. In vitro binding studies indicate a direct interaction between Sec1p and the free pool of the wild-type Sec6p protein, suggesting close interplay between Sec6p and Sec1p in the regulation of SNARE complexes. A coherent model which incorporates all these interactions has continued to be elusive. However, the results I have found do suggest several hypotheses which should prove testable in the future.

Pteridine reductase 1 (PTR1) is an NADPH dependent reductase that catalyzes the reduction of several pterins and folates. The gene encoding this enzyme was originally identified in Leishmania based on its ability to provide resistance to the drug methotrexate (MTX). The DNA and amino acid sequences are known, and overproducing strains of Escherichia coli are available. PTR1 has been previously shown to be required for the salvage of oxidized pteridines (folate, biopterin, and others). Since Leishmaniaare folate and pterin auxotrophes, PTR1 is a possible target for novel anti-folate drugs for the treatment of leishmaniasis. PTR1 catalyzes the transfer of hydride from NADPH to the 2-amino-4-oxo-pteridine ring system yielding 7, 8-dihydropteridines, and to the pteridine ring system of 7, 8-dihydropteridines yielding 5,6, 7, 8-tetrahydropteridines. PTR1 shows a pH dependent substrate specificity. At pH 4.6 the specific activity of PTR1 is highest with pterins, while at pH 6.0 the specific activity of PTR1 was highest with folates. The sequence of PTR1 is only 20-30% homologous to the sequences of members of the short chain dehydrogenase/reductase enzyme family. Although this is typical for members of this enzyme family, it does not allow for unambiguous classification in this family. In fact, when the DNA sequence of PTR1was first determined, PTR1 was classified as an aldoketo reductase. To classify PTR1 definitively, further biochemical characterization was required. To provide this information, the work described here was undertaken: (i) the stereochemical and kinetic course of PTR1 was determined; (ii) residues important in catalysis and ligand binding were identified; and (iii) conditions for the crystallization of PTR1 were developed. The stereochemistry of hydride transfer The use of [3H]-folate, showed that the ultimate product of PTR1 was 5, 6, 7, 8-tetrahydrofolate. 4R-[3H]-NADPH and 4S-[3H]-NADPH were synthesized enzymatically and used as the cofactor for the reduction of folate. PTR1 was coupled to thymidylate synthase (TS), and tritium from 4S-[3H]-NADPH was transferred to thymidylate. Therefore, the pro-S hydride of NADPH was transferred to the si face of dihydrofolate (DHF; see figure I-1). The transfer of the pro-Shydride indicates that PTR1 is a B-side dehydrogenase which is consistent with its membership in the short chain dehydrogenase (SDR) family. The kinetic mechanism of PTR1 When NADPH was varied at several fixed concentrations of folate (and vice-versa) V/K (Vmax/KM) showed a dependence upon concentration of the fixed substrate. This is consistent with a ternary complex mechanism, in contrast to a substituted enzyme mechanism that exhibits no dependence of V/K on fixed substrate. Product inhibition patterns using NADP+ and 5-deazatetrahydrofolate (5dTHF, a stable product analog) were consistent with an ordered ternary complex mechanism in which NADPH binds first and NADP+ dissociates last. However, an enzyme-DHF binary complex was detected by fluorescence. Isotope partitioning experiments showed that the enzyme-DHF binary complex was not catalytically competent whereas the enzyme-NADPH complex was. Measurement of the tritium isotope effect on V/K (T(V/K)) at high and low dihydrofolate confirmed that PTR1 proceeds via a steady state ordered mechanism. Rapid quench analysis showed that dihydrofolate was a transient intermediate during the reduction of folate to tetrahydrofolate and that folate reduction is biphasic. Catalytic Residues of PTR1 The amino acid sequences of dihydropteridine reductase and 3-α, 20-β, hydroxy steroid dehydrogenase were aligned to that of PTR1. Based on the results of the alignment, site directed mutagenesis was used to investigate the role of specific residues in the catalytic cycle of PTR1. Variant enzymes were screened based on their ability to rescue a dihydrofolate reductase (DHFR) deficient strain of E. coli. Selected PTR1 variants (some complementing and some non-complementing) were purified and further characterized. Tyrosine 193 of the wild type enzyme was found to be involved in the reduction of pteridines, but not in the reduction of 7, 8-dihydropteridines, and eliminated the substrate inhibition of 7, 8-dihydropteridines observed with the wild type enzyme. Both PTR1(K197Q) and PTR1(Y193F/K197Q) had decreased activity for all substrates and low affinity for NADPH. In contrast to the wild type enzyme, NADPH displayed substrate inhibition towards PTR1(K197Q). All PTR1(D180) variants that were purified were inactive except for PTR1(D180C), which showed 2.5% of wild type activity with DHF. The binary complexes of PTR1(D180A) and PTR1(D180S) with NADPH showed a decrease in affinity for folate. Based on the kinetic properties of the PTR1 variants, roles for Y193, K197, and D180 are proposed. In conjunction with D180, Y193 acts as a proton donor to N8 of folate. K197 forms hydrogen bonds with NADPH in the active site and lowers the pKaof Y193. D180 participates in the protonation of N8 of folate and N5 of DHF. Crystallization of PTR1 and PTR1-ligand complexes The crystallization of PTR1 from L. major and L. tarentolea as unliganded and as binary and ternary complexes was attempted. Several crystal forms were obtained including L. major PTR1-NADPH-MTX crystals that diffracted to ~ 3.2 Å resolution. It was not possible to collect a full data set of any of the crystals. At their current stage, none of the crystal forms is suitable for structural work.