A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.

Dictyostelium discoideum makes multiple developmentally regulated lysosomal cysteine proteinases. One of these, a lysosomal enzyme called proteinase I, contains a cluster of GlcNAc-␣-1-P-Ser residues. We call this phosphoglycosylation. To study its function, a cDNA library from vegetative cells was screened, and two novel cysteine proteinase clones were characterized (cprD and cprE). Each of them has highly conserved regions expected for cysteine proteinases, but unlike any other, each has a serine-rich domain containing three distinct motifs, poly-S, SGSQ, and SGSG. cprD and cprE cDNAs were overexpressed in Dictyostelium and the active enzymes identified. cprD codes for a protein of approximately 36 kDa (CP4), which is recognized by monoclonal antibodies against GlcNAc-1-P and fucose. cprE corresponds to a 29-kDa protein, which is recognized by antibodies against GlcNAc-1-P. mRNA for both enzymes is present in the vegetative phase and increases during growth on bacteria but decreases throughout development. When the formation of the fruiting body is complete the mRNA for both messages is detected again but in very low levels. Having cloned cDNAs for proteins that carry GlcNAc-1-P should allow us to probe the function of the carbohydrate in these putative lysosomal enzymes.Dictyostelium discoideum is an eukaryotic amoeba that grows as single cells, but when the bacterial food source is removed, the cells initiate a complex multicellular developmental program. Cells aggregate and differentiate into several different types and, in the end, 85% of them are converted into spores setting atop a cellular stalk (1). We are interested in studying the role of carbohydrate modifications in this organism (2). One of these is the addition of GlcNAc-1-P to serine residues, which has been well documented to occur on a cysteine proteinase called proteinase I found in vegetative cells (3-5). Although antibodies against GlcNAc-1-P recognize various proteins in the cells and in secretions of cells grown in axenic medium 1 the identity of these proteins is unknown. To study the function of GlcNAc-1-P on a defined protein, we decided to clone members of the cysteine proteinase family expressed in vegetative cells.Previous studies in Dictyostelium identified two developmentally regulated members of this gene family, cprA (CP1) and cprB (CP2) (6 -8), but none have been identified in vegetative cells. Since cysteine proteinases are highly conserved in all eukaryotes, we used the active site consensus sequence of cysteine proteinases and the cprA and cprB cDNAs to clone two novel vegetative cysteine proteinases, cprD and cprE. They have the predicted conserved regions but also have an unusual serine-rich domain not previously found in any known cysteine proteinase that could be the site of GlcNAc-1-P addition. The cDNA clones were overexpressed and the active enzymes were shown to have GlcNAc-1-P. EXPERIMENTAL PROCEDURESMaterials-Radionucleotides were purchased from DuPont NEN and ICN Biomedicals, Inc. The random primed labeling kit an...

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