Rat liver nucleoside diphosphate kinase (NDPK) and PC12 cell cytosol were used to determine whether NDPK could function as a protein kinase. NDPK was phosphorylated on its catalytic histidine using [␥-

Nm23 genes, which encode nucleoside diphosphate kinases, have been implicated in suppressing tumor metastasis. The motility of human breast carcinoma cells can be suppressed by transfection with wild-type nm23-H1, but not by transfections with two nm23-H1 mutants, nm23-H1 S12OG and nm23-H1 P96S . Here we report that nm23-H1 can transfer a phosphate from its catalytic histidine to aspartate or glutamate residues on 43-kDa membrane proteins. One of the 43-kDa membrane proteins was not phosphorylated by either nm23-H1 P96S or nm23-H1 S120G , and another was phosphorylated much more slowly by nm23-H1 P96S and by nm23-H1 S120G than by wild-type nm23-H1. Nm23-H1 also can transfer phosphate from its catalytic histidine to histidines on ATPcitrate lyase and succinic thiokinase. The rates of phosphorylation of ATP-citrate lyase by nm23-H1 S120G and nm23-H1 P96S were similar to that by wild-type nm23-H1. The rate of phosphorylation of succinic thiokinase by nm23-H1 S120 was similar to that by wild-type nm23-H1, and the rate of phosphorylation of succinic thiokinase by nm23-H1 P96S was about half that by wild-type nm23-H1. Thus, the transfer of phosphate from nm23-H1 to aspartates or glutamates on other proteins appears to correlate better with the suppression of motility than does the transfer to histidines.Nm23 genes have been implicated in the control of tumor metastasis (1). For many, but not all, types of tumors, there is an inverse relationship between the level of nm23 expression and metastatic potential (reviewed in ref.2). Overexpression of nm23 in highly metastatic murine melanoma cell lines (3-6), rat mammary adenocarcinoma cells (7), and human breast carcinoma cells (4,8,9) reduces their metastatic potentials, indicating that nm23 genes can suppress tumor metastasis. Increased levels of nm23 expression also have been associated with both cellular proliferation (10) and differentiation (11). In human tissues the two major forms of nm23 are nm23-H1 and nm23-H2. Expression of nm23-H1 has been correlated with suppression of metastasis (2).The abnormal wing disc (awd) gene of Drosophila encodes a protein that is 78% identical to the human nm23 proteins (12). The awd gene is essential for normal Drosophila development (13). The killer of prune mutation in this gene (awd kpn ) causes a dominant lethality but only in flies that do not have a functional prune gene. The prune gene has been cloned, but the biochemical function of the resulting protein is not known (14,15).Nm23 and awd genes encode nucleoside diphosphate kinases (NDPKs) (12). NDPKs catalyze the phosphorylation of nucleoside 5Ј-diphosphates to triphosphates (16). In the first step, a phosphate is transferred from a nucleoside 5Ј-triphosphate to a histidine at the catalytic site of the enzyme. This high-energy phosphate then is transferred to a phosphate group on a nucleoside 5Ј-diphosphate.The synthesis of nucleoside triphosphates does not appear to explain the role of nm23 in suppressing metastasis (2). Similarly, the effect of the awd kpn mutation...

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The nature of the rate-limiting transition state at zero denaturant (TS(1)) and whether there are hidden intermediates are the two major unsolved problems in defining the folding pathway of barnase. In earlier studies, it was shown that TS(1) has small phi values throughout the structure of the protein, suggesting that the transition state has either a defined partially folded secondary structure with all side chains significantly exposed or numerous different partially unfolded structures with similar stability. To distinguish the two possibilities, we studied the effect of Gly mutations on the folding rate of barnase to investigate the secondary structure formation in the transition state. Two mutations in the same region of a beta-strand decreased the folding rate by 20- and 50-fold, respectively, suggesting that the secondary structures in this region are dominantly formed in the rate-limiting transition state. We also performed native-state hydrogen exchange experiments on barnase at pD 5.0 and 25 degrees C and identified a partially unfolded state. The structure of the intermediate was investigated using protein engineering and NMR. The results suggest that the intermediate has an omega loop unfolded. This intermediate is more folded than the rate-limiting transition state previously characterized at high denaturant concentrations (TS(2)). Therefore, it exists after TS(2) in folding. Consistent with this conclusion, the intermediate folds with the same rate and denaturant dependence as the wild-type protein, but unfolds faster with less dependence on the denaturant concentration. These and other results in the literature suggest that barnase folds through partially unfolded intermediates that exist after the rate-limiting step. Such folding behavior is similar to those of cytochrome c and Rd-apocyt b(562). Together, we suggest that other small apparently two-state proteins may also fold through hidden intermediates.

T4 lysozyme has two easily distinguishable but energetically coupled domains: the N and C-terminal domains. In earlier studies, an amide hydrogen/deuterium exchange pulse-labeling experiment detected a stable submillisecond intermediate that accumulates before the rate-limiting transition state. It involves the formation of structures in both the N and C-terminal regions. However, a native-state hydrogen exchange experiment subsequently detected an equilibrium intermediate that only involves the formation of the C-terminal domain. Here, using stopped-flow circular dichroism and fluorescence, amide hydrogen exchange-folding competition, and protein engineering methods, we re-examined the folding pathway of T4-lysozyme. We found no evidence for the existence of a stable folding intermediate before the rate-limiting transition state at neutral pH. In addition, using native-state hydrogen exchange-directed protein engineering, we created a mimic of the equilibrium intermediate. We found that the intermediate mimic folds with the same rate as the wild-type protein, suggesting that the equilibrium intermediate is an on-pathway intermediate that exists after the rate-limiting transition state.